text:
Function of carapace
Majority of crustacean larvae have expanded carapace drawn out into processes as an adaptation for :
a. Protection from enemies
b. Planktonic habit
The answer given is b but I think it should be (a) as carapace functions as an exoskeleton.
Please help!

I don't know anything about this topic, but surely the key to this is that the question is actually asking about processes extending from the modified carapace, so this is function over and above exoskeleton function. I can imagine that such processes could help with dispersal in currents.

Preface:

Any question about adaptation on macro-level has very little meaning and no precise answer.
There are two types of larvae in invertebrates: primary ("original") and secondary (evolved from post-larval stages). Not digging into the details, nauplius (together with metanauplius) is the only type of primary larvae in Crustacea and it never has a carapace.
"Majority" is by far too strong term, even if we are talking about all larval types. If we just take the largest crustacean orders with species numbers > 1000, together comprising 92% of crustacean species (species numbers are from Ahyong et al, 2011, they seem to be largely correct):

Decapoda - 14.7 k species - rarely have nauplius, marine forms do have planktonic carapaced larvae (principally - zoea)
Isopoda - 10.7 k - no carapace
Amphipoda - 9.9 k - no carapace
Podocopida - 6.2 k - no carapace
Harpacticoida - 6.0 k - no carapace
Cyclopoida - 4.2 k - no carapace
Calanoida - 3.1 k - no carapace
Siphonostomatoida - 2.5 k - no carapace
Cumacea - 1.5 k - have carapace, but development is direct
Mysida - 1.2 k - have carapace, but development is direct
Tanaidacea - 1.1 k - have carapace, but development is direct

So, "majority" could be applied to Decapoda only.
Fabula:
If we substitute "adaptation" with "main biological function" (as you have done) or more correctly "main physiological role":
I would prefer the (a), because carapace does function as a protective structure in those decapod larvae which don't rely on behavioral anti-predatory defenses: see Morgan: "Fishes quickly learned to avoid spined prey, which ... may also increase the rate of evolution of the character". Non-carapaced larvae rely principally on behavior only.
The only contribution of carapace to planktonic life-style in zoea is participation of its spines [when they are pronounced] in parachuting (Chia et al).
[The above should be largely true also for the cypris larva of the cirripeadians, which are enclosed in a non-spined bivalve carapace.]
From the morphological point of view, [at least in adult forms of decapods] carapace creates a chamber for gills, so a morphologist would argue that this is the "main function" of carapace.


text:
Why insects are so small compared to mammals?
I was wondering what biological limitations make the dimension of the insects small compared to the dimension of the mammals. I know in other eras insect were bigger!

Fun: http://science.discovery.com/creatures/10-giant-bugs.htm

@terdon Mothra is that I was searching! Thanks a lot! ;-)

The standard answer to this question is - it's down to the delivery of oxygen to respiring tissues. Insects depend upon a process that is essentially diffusion-driven (via spiracles and the tracheal system) whereas vertebrates have a circulatory system that allows oxygen absorbed in the lungs to be carried via the blood to peripheral tissues. And of course the blood contains specialised carrier cells (red cells) which in turn are packed with haemoglobin which acts as a molecular carrier for oxygen.
The giant dragonflies of the Carboniferous (Meganeura) existed at a time when oxygen levels in the atmosphere were much higher than now (32.5% vs 20.9%), so the diffusion-driven gas exchange system could support a larger body size.

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text:
Electronic laboratory notebook (ELN)
I have been using old style lab book for some time now but with increasing work on computer and storing sequencing results and gel pictures on computer it would be nice to have everything on computer in a form of labbook.
Does any of you uses a good ELN that would be free as I don't think university will be willing to pay for it :)
Thank you for your answers.

I still prefer the good old[tm] Labnotebook. No dependencies on (proprietary) software or a computer. I generate quite some digital data (blots, sequencing etc.) this is stored on the servers of my university. We looked into this in our lab, but discarded the idea pretty fast. Most solutions are either expensive and/or proprietary. Then a lot of these programs are not bought, but rented for a monthly or yearly fee, so once you are in you basically will not get out there anymore. Or it takes a lot of work.
I found one free (for up to three projects), this is Labguru.
There is an article about this on Bitesizebio, which has a few interesting comments. And there is another blog entry about this worth reading.
One of the problems with ELN is a legal one, since a labbook is a legal document providing evidence a what has been done and the results of it. They can be very important when it comes to patent legal battles.

I do not understand the legal problem here. So if I use it data that I put in my ELN might be partly owned by the company who is providing ELN or what?

No, not that. But your entries have to be safe against manipulations. A bound book has numbered pages to prevent the removal of pages with non-fitting results. And it will probably be pretty difficult to move from one solution to another, when a product gets discontinued.

Although there are some definite advantages to physical labboos, and perhaps some legal obligations, I have been loosely using Synbiota for keeping track of general procedures to be done. It has a long list of interesting features, but I particularly like the integrated DNA editing tool, as well as the ability to share projects and updates with labmates.

I am not sure what you need.
Probably Libre Office (a free MS Office like program, including a rich text editor, with image support) will suffice for you.

This is not a decision you, or even your lab can make on its own.  Notebook policies are almost certainly developed by the lawyers of your university.  It would be up to them to certify any change to those policies, in coordination with IT, who would have to be on board to guarantee the integrity of the electronic data.

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text:
DNA fingerprinting
I would like to make my own DNA fingerprint - just for fun to have my "autoportrait":).
I was looking around a bit and all the commercial kits you can have are very expensive. Can you suggest me a cheaper way to do it - which DNA extraction protocol do you recommend?
I have access to restriction enzymes, we have several different pcr polymerases (Herculase, pfu). Can this be used to make PCR with human DNA?
I found this list with primers for different STR loci. http://www.cybertory.org/resources/CODIS/#Primers
If I understand it correctly I have to extract DNA, digest it with restriction enzyme and make a pcr with each of primer pair and than load it to a gel and make a picture. Am I getting it right?
Thank you for your answers!

You might be interested by this Ted talk

What about SNPs instead of fingerprinting or STRs? There are some companies which perform genotyping at hundreds of thousands of loci for acceptable money.

The idea is that I do it by my self not just send DNA to some company.


text:
Generalism - specialism index in microbial ecology
I am doing research in Lipid metabolism in different yeast species and I would like to look into ecology of them.
I would like to classify them to a Generalist/specialist based on different Parameters (growth on carbon sources, pH, temperature,...).
I wonder if you have any experiences with that classification and if any of you know which indexes are used for that in microbial ecology?
Thank you very much for your answer!
Martin

Can you expand a little on your classification desires, you're not very specific.  There has been plenty of research on lipid metabolism, including in yeast.

I've published a paper comparing different measures of specificity, if that is of any help. But if you plan to have, e.g., measures of performance on carbon sources, pH, and temperature, then you'll have three measures of specificity (i.e. one for each).
Also, worth to have a look at Graham Bell's inconsistency and responsiveness, as used by Venail et al. to classify specialists and generalists.


text:
Gibson assembly - primer design with A and T rich regions
I have question about Gibson assembly. I have done it several times and it always worked okay for us, but now I want to assemble together a fragment which has sequence like this: 5'CTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTT3'.
Do you think a AA and TT repetitions will cause any problems? We usually used 30 bp of homology at both sides of sequence. Should I use more in this case? What are your experiences regarding this AT repetitive sequences?
thanks,
Martin Kavšček

Yes this is a very low-complexity sequence. Can you paste your entire sequence? There might be some trick to work it out.


text:
Multiple clones Sanger sequencing analysis
I am doing a 96-well arrayed cloning. I have done Gibson, transformations and minipreps in a 96-well format, then sent out the plate for sequencing. Normally, I sequence 3-4 clones and check if the sequencing looks good manually with Benchling cause it has nice visualization tool that highlights mismatches but also displays sequence quality. Now I have to check the sequences of 96 individual clones. Obviously I can write a simple script that would do alignments and report the alignment quality. However, I am guessing there should be already tools available that work with Sanger clonal sequences and provide nice visualization for many alignments at once. Any recommendations for specific tools I should use?
Thank you!

I haven't personally used it but seqtrace seems to be targeted for this purpose. It seems to be able to do alignments and facilitates multiple trace file analysis.
I worry a little bit about not visually inspecting trace files for sequencing of clones, but I may be a relative dinosaur (about sanger at least).
Here is the abstract:

Modern applications of Sanger DNA sequencing often require converting a large number of chromatogram trace files into high-quality DNA sequences for downstream analyses. Relatively few nonproprietary software tools are available to assist with this process. SeqTrace is a new, free, and open-source software application that is designed to automate the entire workflow by facilitating easy batch processing of large numbers of trace files. SeqTrace can identify, align, and compute consensus sequences from matching forward and reverse traces, filter low-quality base calls, and end-trim finished sequences. The software features a graphical interface that includes a full-featured chromatogram viewer and sequence editor. SeqTrace runs on most popular operating systems and is freely available, along with supporting documentation, at http://seqtrace. googlecode.com/.© 2012 ABRF.

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text:
Drugs with unknown mechanism of action
I want to get a list of FDA-approved drugs with unknown mechanism of action. I think that there should be a database where for each drug there is a short field "mechanism", but I haven't find one. Do you know any database where I can find such information? Another possible way is to use text-mining programs to analyze scientific libraries like PubMed and find the drugs which names often occur together with words "mechanism" and "unknown". Are there any good programs which can do it? Any other suggestions are welcome too, of course.

There are a lot! Most drugs bind many targets, and nobody knows for sure which targets are responsible for the drug action. For example, look at wide-used drug modafinil (http://www.drugbank.ca/drugs/DB00745)


text:
can recessive alleles produce codominance?
Maybe I'm confused by the term "codominance", but I wondering if codominance only occurs with two dominant alleles.
Can two recessive alleles produce codominance in an individual?
Likewise, can two recessive alleles produce incomplete dominance?

An allele is not dominant or recessive by itself. It is dominant or recessive compared to another allele. Therefore, if you consider one locus (position on a sequence) that has two alleles (bi-allelic locus), you cannot have two dominant or two recessive alleles. It is like saying that two things are darker. In reality you can either say that one is darker than the other one or that the color is as dark as the other one. So either one allele is dominant (or partially dominant) and the other is necessarily recessive (or partially recessive) or the two alleles are codominant.

Think of a gene that influence a phenotypic trait such as tail length for example. This locus is bi-allelic (two different alleles (=variant of a gene) exist (=segregate) at this locus). We'll call the two alleles $A$ and $B$. An individual that homozygous $AA$ has trait value $M_{AA}$. The homozygotes $AB$ have trait value $M_{AB}$ and individuals $BB$ have trait values $M_{BB}$. Viewing this in a table it gives:
$$
\begin{array}{c|lcr}
\text{Genotype} & \text{Tail length} \\
\hline
\text{AA} & M_{AA}\\
\text{AB} & M_{AB}\\
\text{BB} & M_{BB}\\
\end{array}
$$
The following table gives the tail length of the different genotypes under the different types of interactions (dominance, partial dominance and co-dominance).
$$
\begin{array}{c|lcr}
\text{Interaction} & \text{AA} & \text{AB} & \text{BB} \\
\hline
\text{A is dominant and B is recessive} & M_{AA} & M_{AA} = M_{AB} & M_{BB} \\
\\
\\ & & M_{AA} > M_{AB} > M_{BB}\\
\text{Partial Dominance} & M_{AA} & \text{or} & M_{BB}
\\ & & M_{AA} < M_{AB} < M_{BB} \\
\\
\text{Codominance} & M_{AA} & M_{AB} = \frac{M_{AA} + M_{BB}}{2} & M_{BB} \\
\end{array}
$$
Because, in the codominance case $M_{AB}$ is necessarily between the values of $M_{AA}$ and $M_{BB}$ ($M_{AA} > M_{AB} > M_{BB}$ or $M_{AA} < M_{AB} < M_{BB}$), one can consider codominance as being a special case of partial dominance where neither $A$ or $B$ dominates because they both contribute as much to the determination of the tail length.
Various definitions may exist for such concepts as well as for overdominance (also sometimes called heterozygous advantage) or stabilizing selection for examples. Those definitions differ in the sense that they sometimes apply to the value of the quantitative trait, to the fitness value (which is another quantitative trait) or to genetic polymorphism.

When two alleles show codiminance, they are not described as dominant or recessive relative to each other. They are simply codiminant to each other. The same applies to incomplete dominance.
Note that all these terms are relative to the alleles you're talking about. An allele that is dominant over one allele may be recessive to another and codiminant with yet another.

Shorter answer:
The dominant/recessive paradigm that is taught in middle school and high school is not universally applicable.  It accurately describes only a small number of phenotypes, compared to all the genotype/phenotype interactions which an organism exhibits.  It was a useful place to start when it came to unraveling the relationship between genes and phenotypes, but it is very limited.
So trying to shoehorn all phenotypes into that paradigm is a serious mistake.  Just because all of your homework problems are solved using it doesn't mean that it's applicable to all real-life situations.


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Role of Melatonin in Circadian Rhythm
Melatonin regulates the wake-sleep cycle by causing drowsiness and lowering body temperature, but which specific chemical pathways lead to this drowsiness?

This is a quite complicated question, as melatonin has quite a range of effects on the cells. First, melatonin binds to three different melatonin receptors, which are found in a wide variety of tissues or organs including brain and retina, cardiovascular system, liver and gallbladder, intestine, kidney, immune cells, adipocytes, prostate and breast epithelial cells, ovary/granulosa cells, myometrium, and skin.
Two of the receptors a G-protein coupled receptors (GPCR), the MT1 and MT2 receptors. The third is a quinone reductase called QR2.
For more details, see this papers:

Melatonin receptors in humans: biological role and clinical
relevance.
Melatonin receptors and their regulation: biochemical and structural
mechanisms.

The role of melatonin is pretty broad, see the figure (from this article:"Melatonin: Nature's most versatile biological signal?")

The receptors transport the outside chemical signal (the melatonin) to the inside and activate (or repress) a number of different signal transduction pathways. See the figure below (taken from Qiagen):

This includes MAP-Kinase pathways, proteinkinase C, IP3, adenyl cyclase and so on. This is quite complicated and I recommend reading some of the following articles if you are interested in a deeper insight. Finally these signals are transmitted into the nucleus and result in a changed gene regulation/expression.

Melatonin receptors, heterodimerization, signal transduction and
binding sites: what's new?
Physiological effects of melatonin: role of melatonin receptors and
signal transduction pathways.

The second picture is beautiful !

From wikipedia article on melatonin receptors (with some modifications) :

In mammals, melatonin receptors, which are G protein coupled receptors, are found in the brain and some
  peripheral organs. However, there is considerable variation in the
  density and location of MT receptor expression between species.
MT1
In humans, The MT1 subtype is expressed in the pars tuberalis of the
  pituitary gland and the suprachiasmatic nuclei of the
  hypothalamus....This is indicative of its role in circadian and
  reproductive function.
MT2
The MT2 subtype is expressed in the retina. MT2 receptor mRNA has not
  been detected by in situ hybridization in the rat suprachiasmatic
  nucleo or pars tubealis. This receptor may have a role in light
  dependent functions.

I know this does not tell about the pathway but I suggest you try searching some of the papers cited at the end of the wikipedia article.

Oh, I completely missed this topic. It is interesting and I will have a look into thi later this evening.


text:
Mechanisms of Flagella
I know that dynein arms and microtubules are involved in the contraction of certain parts of the flagella to produce a wave motion, but I don't understand they are related to the 9+2 structure of the basal body.
How do flagella work in prokaryotes and eukaryotes?

Flagella in prokaryotets is very different from that of eukaryotes.
The main structural protein in a prokaryotic flagellum is flagellin. The flagellar motion is caused by a molecular motor similar to Mitochondrial ATP-synthase; molecular motion generated by the chemiosmotic energy.
Eukaryotic flagella or cilia are made of microtubules with dynein attached on to them and cause the movement by the energy generated by ATP hydrolysis.
See the wikipedia article. It is clear enough.


text:
How does a prophage leave the host cell's genome?
I understand that, unlike a prophage, a provirus never leaves the genome, but I don't understand how the prophage "leaves".

The prophage is usually activated by cellular stress (bad nutrient conditions, toxic environment and so on) or cell damage.

This is explained reasonably well at the Wikipedia entry for phage λ, which is the prototype lysogenic phage. Basically, integration occurs by site-specific recombination between the attP region on the phage genome and the attB region in the host genome. This means that in the prophage form the phage DNA is flanked by direct repeats. Excision involves the reverse process - recombination between these direct repeats. Excision is promoted by the phage-encoded Xis and Int proteins in combination with the host protein Ihf. The regulatory cascade which links cellular stress to this recombination event is detailed at the linked page.


text:
Anaphase Microtubule Disassembly
Centromeres move toward the poles as the microtubules that connect them shorten. This shortening is due to the removal of tubulin subunits from the kinetochore ends of the microtubules.
I'm confused as to how the centromeres can be pulled closer to their respective poles while tubulin subunits are being removed. To me, this is similar to pulling an object by a rope while simultaneously cutting pieces of the rope out. How do the centromeres remain connected to the spindles while tubulin subunits are being removed?

I don't think the microtubules shorten. The disassembly of tubulin (dynamic catastrophe) happens from its + end which in this case is protected by the kinetochore proteins. The separation of chromatids, I guess, happens via a dynein (and in some cases kinesin) mediated movement.
The wikipedia article on kinetochore is quite detailed and you can have a look at it.

Sorry, I was unclear. I meant that the length of the microtubule spindle fibers shorten due to the removal of tubulin subunits.

yes.. but it does not happen thay way as I can recall. the removal of tubulin subunits happens from the + end which is now protected.

How exactly do the kinetochore proteins protect the + end?

In different species it's different - in some it's packman like ie disassembly from the + end but in some the microtubules are reeled in (from the centriole end)

I know this question was asked a very long time ago, but for future viewers, below is a useful snapshot taken from my Campbell's Biology Textbook. As you can see, the motor proteins are not located at the very "tip" of the microtubule, so they are still able to carry the chromosome to the pole while the tubulin subunits disassemble on the kinetochore end. For more about motor proteins, go here, to YouTube, or wikipedia.


text:
How likely is it that Earth has millions of different species?
I know that the probability of life existing on a habitable planet is pretty slim. But, how likely is it that it hapenned millions of times in the same planet? Or was it only a few species that evolved into millions of other different species over time?

Hello! I am not sure I understand your question. Do you mean: "How many times did life originated de novo (from non-living things) on earth?". If this is your question, you might want to change your title which is misleading. We have the feeling that you ask about how many species exist today.

I mean if life is  very rare to happen spontaneously how do we have millions of different species?

Because of speciation (evolutionary process by which one species becomes two!). Would something like this answer your question?

We can be reasonably certain that ALL species that exist today share a common ancestor, a common origin. Every single species we have even encountered, for example, uses a long chain of nucleotides (DNA or RNA1) to store its genetic information. If life had arisen through multiple independent sources, we would expect to find different solutions to the problem of passing genes to offspring.
To put it another way, all forms of life we have found can be placed in one of three main classes (superkingdoms): eukaryotes, prokaryotes and archea. All other types of life come under one of those three categories (viruses are special and not really considered alive2):

Now, any organism we have ever seen uses the same basic systems of storing its genetic information. In fact, there are even certain genes that are conserved (similar in sequence) across all domains of life (see my answer here for some examples). That is why this version of the tree has a common root. All lines start from a single original line (commonly known as the Last Universal Common Ancestor, LUCA).
In summary, all the evidence we have points to a single origin of life. There are too many shared features to assume multiple origins. Despite their enormous diversity, all organisms share certain very basic systems.

NOTES

As far as I know, only certain viruses use RNA instead of DNA, all truly living creatures use DNA. Even if I'm wrong about this, the differences between RNA and DNA at this level are small enough that the common origin still holds.
See here for more in why viruses are not alive.

Another proof of the common origin of life is the use of (almost) the same codons for coding proteins. Or that the sequence DNA -> RNA -> Protein is also always the same.

@Chris well, there are actually at least 20 different genetic codes (granted they don't change that much) so I did not want to use that one. Good point on DNA->RNA->Protein though.

I know that there are some differences. But given that the three kingdoms seperated quite a long time ago, its pretty amazing, that we can still translate human proteins in E.Coli and they are functional. And the difference is quite small if you think about the sheer number of species.

@Chris absolutely, I agree. I just didn't want to use it as an argument (though I would if debating another biologist) since there are actually some differences.

Thats a good point. :-)

um, reverse transcriptase, anyone? (biochemistry) A polymerase that catalyzes the formation of DNA from RNA; found in retroviruses.

@Susan, actually found in many species, encoded by repetitive elements like LINES and ERVs etc but in none of them is it used to drive the expression of native protein coding genes nor does it change the fact that the flow of information is from genotype to phenotype via RNA: DNA->RNA->protein.

The fantastic number of species that one can observe today is not a fixed value. This number changes over time. These changes are not caused by De Novo origin of life but by two processes which are:

exctinction

Process by which a species disappear

speciation

Process by which one species (or one lineage) becomes two.

Therefore the absolute change in number of species equals the difference between speciation and extinction.
$$\delta_{species} = speciation.rate - extinction.rate$$
There is a ton to say about extinction and speciation and it would take several books to tell you about this science. If you want to study the subject a bit further, I would suggest you to first grab a book of general biology or an introductory book in evolutionary biology before jumping into a book that talk about speciation and extinction.
One can map how speciations events occurred frmo a unique common ancestor in order to create the diversity of life you can observe today. Such mapping are called Tree of Life. Here are the roots of the tree of life of living things on earth. You can spot Animalia, Fungi and Plants on the far right of the picture. The Common ancestor of all living things on earth is called LUCA (Last Universal Common Ancestor).

As a sidenote: The concept of species itself accept several definitions.


text:
Significance of having goosebumps?
What help does goosebumps do to us? I don't think it helps us noticeably when we are scared or shivering.

As a general rule, you might want to check wikipedia before posting here. On the other hand, I am glad you made me look the answer up.
Goose-bumps warm you up a little. "During the formation of goose bumps, the body is warmed from the muscle tension in piloerection."
There are other reasons, which do not directly serve humans, but may be a remnant from evolution. In animals with fur, the erected hair makes thicker insulation and further protection from cold. "Goose bumps can also be a response to anger or fear: the erect hairs make the animal appear larger, in order to intimidate enemies."
More on wikipedia: http://en.wikipedia.org/wiki/Goose_bumps

Hairs (with their nerve rich follicles) are sense organs, extending our sense of touch beyond the skin. When things disturb our hairs we feel them; bugs, breezes, close encounters. During fright or arousal goosebumps cause the hairs to stand on end, extending our sense of touch to it's furthest distance. In the process the hair shafts that are otherwise laid against each other are separated, reducing the dampening of vibration and movement from shaft to follicle - which increases fine sensitivity. Also during goosebumps the smallest disturbance of hairs causes a reaction in surrounding hairs, amplifying distinguishable sensations into hard to ignore. Goosebumps are sensory enhancers.
This goosbumps response predated hairs as fur for insulation and probably mammals as a group. Raising fur to increase insulation, as well as raising hackles to look intimidating almost certainly piggy backed on this pre-existing response.  It is a function that has never been lost and should not be considered vestigial.

"The formation of goose bumps in humans under stress is a vestigial
  reflex; a possible function in human evolutionary ancestors was to
  raise the body's hair, making the ancestor appear larger and scaring
  off predators. Raising the hair is also used to trap an extra layer of
  air, keeping an animal warm. Due to the diminished amount of hair in
  humans, the reflex formation of goose bumps when cold is also
  vestigial."

This is from the wikipedia article on human vestigiality under Behavioural category.

My guess was that it was used in the past as a technique to scare predators, seeing as our ancestors had a lot of hair, this could've been a lot of use.


text:
Why is the Tm defined as the temperature at which 50% of dsDNA has changed into ssDNA?
In molecular biology, Tm is defined as the temperature at which 50% of dsDNA is converted to its single stranded form. Intuitively it would seem that the melting temperature should have been defined as the temperature at which the entirety(100%) of the dsDNA converts into ssDNA. What is the basis of using this ratio, i.e, 50% in the definition of Tm?

If you search for DNA melting temperature plots you will see they have a sigmoid shape. It's common for those kinds of plots to be described by the 50% value for example the therapeutic index and the 50% lethal dose of a substance (LD50). This is because at really low and really high temperatures all DNA molecules become dsDNA or ssDNA respectively so that information is not really useful. You could maybe talk about the first temperature at which DNA becomes 100% denatured but the curves are highly nonlinear at that region so it's difficult to quantify. The 50% is an easy way to compare different molecules since that region is usually linear and easy to compare in a meaningful way (basically all molecules have the same ends of the sigmoid shape, but it's the middle part that changes the most).

The point to emphasize is that in both sigmoid or rectangular hyperbolic curves (for MM enzymes and many ligand-binding cases) you never actually get to 100% until infinity — the curve approaches it asymptotically. So you can't measure it acurately. You need a mathematical function that can be measured and will differentiate different cases. A figure would help.

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text:
Does our brain really "flip" images perceived by our eyes?
I know a similar question has been asked and this is closely related to cognitive science. However, I want to know whether is there any mechanism or structure in the visual cortex which is proved to be flipping the images upside down.

I recommend you go over to cogsci.se and have a look at this thread. Its basically the same question and the answers are pretty nice including some references.

@Chris.. i wonder why cognitive sciences is a separate SE. It is a part of biology.

@WYSIWYG It is true that many questions asked here on Biology could equally be asked on cognitive science. However, there are many questions in cognitive science that are part of psychology and therefore has nothing to do with biology. How are we supposed to treat a question that is a duplicate on another SE website? Should we close it as off-topic even if it is not really off-topic? The comments are not the right place for having this discussion. Sorry :)


text:
Recalling memories by electrical impulses?
I've read that in an experiment, a pair of stimulating electrodes were inserted into a blind man's visual cortex, and upon passing electricity, the phosphene phenomenon was produced. Is it the same with memory? Can each and every memory, of an individual be recalled by a similar method? And if so, in which region of the brain will the stimulating electrodes be inserted?

As far as I know, we do not know very much about the whole mechanism of memory. My guess is: no. You would be better off trying hypnosis.

You might be interested in reading the June 2013 issue of Scientific American which contained an interesting article on forming of memories..

it is important to first map the memories. Usually a memory involves a lot of different perceptions. So i guess it is not that easy. visual cortex is a specific anatomical location that is involved in a very specific task.

There have been quite a few studies on stimulating the brain with electrical pulses and it has been found to be successful too. Three papers of particular interest I found were:
1) In a study titled "Explaining How Brain Stimulation Can Evoke Memories", it was found that electrical stimulation in the temporal neocortex can cause neurosurgical patients to spontaneously experience memory retrieval. The subject's brain in the study when stimulated enabled him to recollect memories from the time that he was in high school. They concluded that there are patches of neurons encoding memories for differnt periods (in this case the high school). (reference)
2) In the second study titled "Memory Enhancement and Deep-Brain Stimulation of the Entorhinal Area", it was observed that

entorhinal stimulation applied while subjects learned the locations of
  landmarks enhanced their subsequent memory of these locations: and the
  subjects reached these landmarks more quickly and by shorter routes,
  as compared with locations learned without stimulation. (reference)

3) In a third study titled "Stimulation of Entorhinal Cortex Promotes Adult Neurogenesis and Facilitates Spatial Memory", it was found that stimulating a specific region of the brain leads to the production of new brain cells that enhance memory. (reference)
In two on these studies, memory is enhanced by stimulation to the entorhinal cortex and in the first study the neocortex was stimulated. So, in a broad sense the  cerebral cortex is generally used for these studies.
This is a continuous field of research as a treatment to diseases like Alzheimers (reference).

Gray-Brodman-Entorhinal Cortex (Wikipedia)

Neocortex location (reference)


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How would constantly growing nails have aided early human?
Nails grow rapidly and constantly, such that without constant artificial trimming they would reach lengths difficult to manage. How did this benefit early humans, say 200kya? Were they used like claws? Did physical activity keep them at a short length?

My guess is that they would work very much like the claws of other animals. Dogs for example will trim their nails by walking around outside. Dogs that live in houses need to have them cut.

There's lots of reading about chimp and human nails. when you consider all the little things that they are used for, nails are used for very many things, including protection of finger ends, food, combing, contests. The toes do grow less than half as fast, so it seems that the speed is fairly optimal. The answer, however is unkown with precision.

I can't say anything for sure (and, with questions like this, it's rare that anyone can), but my guess would be that human nails grow constantly for the same reason as the (analogous) claws of most mammals do: to keep them from accumulating damage.
Your bones, if they suffer mild to moderate damage, are capable of healing through the concerted action of several specialized cell types in your body.  This healing process, however, is only possible because the bones are inside the body, surrounded and permeated by living tissue.
Your nails, however, are simple sheets of keratin, and contain no living cells that could heal them, nor any blood vessels that could keep such cells alive.  Being exposed to the outside of the body, outside the skin barrier, they cannot really support living tissue that could heal them.
Instead, the way human nails (and animal claws, more generally) "heal" is by growing.  Since the nail itself cannot contain cells that would rebuild it, those cells are instead located in the nail matrix, which is located under the nail and from which the exposed part of the nail continually grows out of.
(Indeed, the same is true of most of the exposed parts of your body: your hair and even the surface of your skin itself are also composed of non-living keratin constantly growing from an underlying matrix.  This is necessary to maintain the skin barrier between the interior and the exterior of the body.)
The reason human nails must keep growing all the time is that they do continually wear down and suffer damage from various causes (and would surely have worn down much faster yet for our primitive ancestors, who had to sustain themselves through hard manual labor with few or no tools).  If your nails just stopped growing after you reached adulthood, they'd be in horrible shape even just a few years down the road, not to mention decades later.
While the rate at which human nails do grow is pretty slow — it takes several months for a damaged fingernail to fully regrow, and up to a year for a toenail (a fact I can confirm from personal experience, having broken my toenail last summer) — it's still enough to ensure that your nails will only bear less than a year's cumulative damage at any moment, rather than every nick and scratch and cut they've suffered during your entire lifetime so far.
In a relatively sheltered modern lifestyle, it's quite likely that even this moderate growth rate may exceed normal wear on your nails, requiring them to be clipped regularly.  Still, the fact that you can clip your nails (or, in a pre-modern society, just bite or scrape or cut them down) reduces the selection pressure against excessive nail growth.  On the other hand, not having your nails grow back as fast as you wear them down through hard manual work could be quite bad for your fingers, and could easily reduce your working ability or even lead to harmful infections.  Thus, it makes sense that evolution has optimized out nails to grow at a rate that, on average, tends to somewhat exceed the typical rate of wear.

Good answer +1. That'd be great if you could highlight that the possible reason humans have nails that grow indefinitely is that due to phylogenetic constraint and absence of strong enough negative selection. This will clarify the reason why you mainly answer to  "why do mammals claw grow indefinitely".

@Remi.b: That's not really what I'm claiming here. My actual claim is that, even for humans, there is selection pressure to keep nails growing constantly: if your nails stopped growing at, say, age 20, they'd probably be all but gone by 30 simply due to normal wear and tear. Whether the rate at which human fingernails grow is optimal for a modern urban lifestyle is another question, and one that is harder to answer definitively. But for that I do argue that, if there's any net selection towards slower nail growth (which I'm not totally convinced of), it's surely weak and of recent origin.

Oh..right. sorry about that, I read your post too quickly, that is indeed what you are saying. ok.

contrast the nails as described above with tooth degradation. Adult human teeth does not regenerate or grow

It didn't have to benefit them, evolution has no intent and not all traits are advantageous. Not every thing we have in our bodies is there "for a reason", some things are just there, others evolved, and some are vestigial (inherited from our ancestors).
A whale can move his tail up-down only because he's a mammal and can't ever be anything else, while fish move their tails left-right because they're fish and that's their heritage.
Constantly growing nails never helped a single human, but they sure did help our ancestors which had claws and needed them for survival. We lost the claws but didn't lose the constantly growing trait. Basically, next time you trim your nails remember they (in shape of claws) saved your ancestor's life a long time ago and that's why you have them although you don't need them. He needed them.

I don't think this is a very good answer. It sounds like you're arguing that the constant growth of human nails is just a useless vestigial trait. Yet the change from claws to flat nails in our evolutionary history is a non-trivial one, and must surely have involved some considerable selection pressure. Had it been advantageous at the time for our nails not to grow during adulthood (or at all!), that, being a comparatively minor change, surely could've evolved as well.

The change from claws to nails happened when we became monogamous (bipedalism evolved around this time as well) and could therefore expend resources into something else than fighting each other for females. That's how we lost the jaw muscles and allowed for brain growth, and that's when we lost canine teeth as well. There was no selective pressure to lose those traits, but the absence of one. Rival males. That's why we don't have claws, but still have evidence of having them a long time ago.

Also, "advantageous" or "deleterious" is only important when it affects your ability to survive long enough to reproduce. If it doesn't, there is no selective pressure on that trait even if it's deleterious. The reason for the change from claws to nails is energy - it was better used elsewhere. Bear in mind that hunger is prevalent in the wild, and organisms which had smaller claws were more likely to not die of starvation. Or/and were better in tool use. In any case, when claws got small enough the selective pressure disappeared and that's why we didn't lose them completely.

An interesting theory, but I'm not sure how it can explain the fact that all great apes have essentially human-type nails, as do most other primates. While some primates are indeed more or less monogamous, many are not; this includes our closest evolutionary relatives, the chimpanzees and bonobos, which nonetheless have very human-like nail morphology.

You're right, I overlooked that fact. The development of nails must have taken place much earlier. The most recent common ancestor of us and great apes must have had nails already, otherwise we'd be saying nails evolved separately in each species. No fossils of that ancestor have been found and we're down to speculation about the time when it lived and it's physical traits.


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Why are oceans carbon sinks?
Building on this question,
I understand pretty clearly why forests are carbon sinks (trees grow, and there is carbon stored in soil and biomass).
What about the oceans, though? Are underwater plants the real actors here?

https://en.wikipedia.org/wiki/Carbon_cycle#/media/File:Carbon_cycle.jpg
Look at the white number in parentheses labelled "deep ocean". Solubility is the biggest actor of them all

@Luigi You should make that an answer.

Check out the excellent Wikimedia picture of the carbon cycle:

All the numbers are in billions of tons of carbon: white = stored, yellow = natural flux, red = human contribution. Notice that the deep ocean stores much more accessible carbon than any other carbon cycle source, and is only surpassed by the lithosphere* in overall quantity of carbon stored on the planet. Some is stored by algae/sea plants, but the vast majority is stored as dissolved CO2.
*The lithosphere (crust and upper mantle) is not involved in the carbon cycle, and is therefore not shown in the above image.

The diagram in the answer by @Luigi does a great job describing how oceans operate as both short-term and long-term carbon sinks. Where phytoplankton can immediately uptake carbon and then upon their demise (as well as the demise of other marine organisms), the action of their sinking to the ocean floor transmits carbon to the deep ocean, where carbon is stored for decades to centuries (based on where in the ocean this occurs).
The second question you ask about underwater plants is especially interesting and an area of active research! For example, the Blue Carbon Initiative is trying to evaluate the global potential of "blue carbon ecosystems", that is coastal photosynthetic systems (i.e., Mangroves, Salt Marshes, and Seagrasses), to sequester carbon in their soils.
In addition to carbon seqeuestration, there is also the problem of ocean acidification, where the absorption of CO2 in seawater is making the ocean more acidic. Under those circumstances, both seagrasses and kelp forests are being considered as potential "ocean acidification refugia".
And, the state of California recently passed legislation to understand the role of eelgrass, a specific type of seagrass, in sequestering carbon and reducing the impacts of ocean acidification.


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Can someone identify this bird? It looks like it has a few deformities

All pictures taken at Lake Roberta in Tampa, FL.
All pictures depict the same bird.
(The wing deformities can be explained by the lake water being slightly highly toxic, but the neck I can only assume to be caused by the Muscovy ducks that live in the lake.)

What does "slightly highly toxic" mean? Just a little bit highly toxic? Sort of toxic? Mildly polluted?

How can ducks make it have an odd neck? What's odd about it?

It's an anhinga, as@kmm commented. There's nothing wrong with it; it's not deformed in any visible (to me) way. They spread their wings to dry, as they are darting birds that do not float on the surface of the water, but beneath it.
Here's one drying itself on a turtle.


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How do you determine if donated, stored blood is viable?
Think about stored donated blood; What should we check before transfusing it to a patient, to see if blood is still viable?
What methods to use?
What components should it have in what levels, to be counted as viable?
Detailed version of the question, including some background:
Since I'm a software engineer I'm not aware which kind of topics should I search to find answers that I need. Of course I have tried to find publications and books related directly to this topic. But I could not find any. What I need at the moment is some material to do "some reading/research". And of course, if possible (which I don't know if there are too many possible answers or not) direct answers would be extremely helpful.
Of course I have done some research on indirectly related topics. For example; I have read papers about results of blood storage on blood quality etc. But these papers just analyze the effects, while it is not clear for me if these analyzed materials represents viability of blood.
For more detail, things those analyzed in few papers that I have read are;

CBC: The CBC consisted of 10 parameters: white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet (PLT) count, and mean platelet volume (MPV).
Differantial: consisted of relative percentages (%) and absolute numbers (#) of neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
Reticulocyte: The reticulocyte enumeration consisted of relative percentage (retic %) and absolute number of reticulocytes (retic #), and immature reticulocyte fraction(IRF).
The NRBC: enumeration consisted of NRBC per 100 WBC (NRBC %) and absolute number of NRBC.
Others: Factors V, VII, VIII, X, and XI, protein S (PS), a2-antiplasmin (AP), and fibrinogen; and the activated partial thromboplastin (APTT) and prothrombin (PT) times. Factors VIII and factor IX.

Still, I'm reading about each of these. What I'm in need is someone help and say; like:

"You are totally lost, these are not related to your case"
"You should check this paper which is related to your case"
"This, this and this ones in your list are what you are looking for. Focus on them."

Thanks

Have voted to reopen. Great job on sharing your research. A question (which will help direct the answer): why do you want to know this? You've listed some of the normal parameters of blood, many of which have nothing to do with viability.

I'll work on new blood testing techniques for viability. While I'll be working with people from health area, first I want to learn about this topic myself, with my researches. I have been looking for sources on such topics but could not find a solid lead.

Blood can be stored as whole blood or packed red blood cells. The constituents of each are different. Which one will be used varies based on the patient, the availability and the situation. Whole blood (not necessarily warm) is used more often when there is massive blood loss, as it contains all the components of blood: clotting factors, platelets, plasma, etc. Packed red blood cells have had the white blood cells removed, most of the plasma, the platelets, etc. It is indicated for routine blood loss and to increase the oxygen carrying capacity of blood. The constituents of each are different.
The primary problems of stored blood viability are red blood cell deformability (red cells are normally very flexible in order to pass through capillaries) and red blood cell fragility (this results in fewer RBCs actually active in the recipient, as RBCs will break apart.) The viability of RBCs in storage depend on factors such as adequate ADP (the energy substrate). When RBSs are less viable for any reason (less deformable, more fragile, or hemolysis has started in the stored blood), this is termed red cell (or red blood cell/rbc) storage lesion.
Though there are probably many questions you may have about storage methods and viability, this will give you an idea of search terms for your reading. Good luck in your research efforts.
Fresh Warm Whole Blood (FWB
2012 Clinical Practice Guide on Red Blood Cell Transfusion
Blood Products
Deformability of stored red blood cells. Relationship to degree of packing
Microvascular perfusion upon exchange transfusion with stored red blood cells in normovolemic anemic conditions
Studies on the preservation of human blood. II. The relationship of erythrocyte adenosine triphosphate levels and other in vitro measures to red cell storageability
Nitric Oxide Scavenging by Red Blood Cell Microparticles and Cell-Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion
Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy


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Are there any good reviews on the mouse and human conservation in coding and gene regulatory module regions?
I was looking for reviews that deal with conservation between mouse and human of proteins, noncoding RNAs, gene regulatory module (such as promoters and enhancers), gene regulatory networks post the mouse genome sequencing era

you can look at the conservation tracks in UCSC genome browser.. or download latest MAF file from there.. You can do the analysis yourself.. You wont find reviews on this specific question.


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what is the difference between early blight of potato and late blight of potato?
apart from different fungus causing these diseases in potato, how are Early Blight of potato and Late blight of potato different?

Some Differences are listed here

Please consider reading these articles (article 1, article 2)


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Beetroot white skin mould
Does a mould make a beetroot poisonous or inedible ?
Lemon's mould for instance, makes penicillin, but it's green there, and I'm allergic to penicillin, would the white mould produce penicillin too?

Owing to the large number of fungal genera, can you please expand on how the mould developed, its characteristics or maybe a picture? That could narrow the question down.

It was over a month in the fridge :D.

To be honest, whichever the answer, better to stay on the safe side and throw it away.

From a study titled "Production of Penicillin by Fungi Growing on Food Products: Identification of a Complete Penicillin Gene Cluster in Penicillium griseofulvum and a Truncated Cluster in Penicillium verrucosum", moulds on food items were analyzed for fungi that could produce penicillin. The conclusion drawn was

Among different fungal species belonging to the genus Penicillium that
  are currently used as starter cultures in the food industry or that
  are frequently isolated from cured meat products, only P. griseofulvum
  (in addition to the previously reported P. chrysogenum and P.
  nalgiovense) was found to produce penicillin and possess the three
  penicillin biosynthetic genes (pcbAB, pcbC, and penDE).

Even penicillin should be avoided as the paper states that

The presence of penicillin in food must be avoided, since it can lead
  to allergic reactions and the arising of penicillin resistance in
  human-pathogenic bacteria.

During world war two, a strain of Penicillium chrysogenum was isolated from the mould growing on a cantaloupe (reference) for the development of penicillin. This was after searching through many mould families developing on different food items so penicillin strains do develop but are rare.
The problem faced is that moulds cannot be differentiated from each other through sight nor is all penicillin moulds safe (reference). Whatever be the case, I hope that you didn't venture to try and eat that beetroot.

Actually, I ate it :D, and it's been like 6 months, and I'm still alive :).

I can come up to a conclusion that I need to retest my self for penicillin, cause I went through some body enchantments, I doubled my immune system, perhaps I'm no longer allergic to it.

@That is debatable because there may not have been any penicillin producing moulds at all. Getting a retest is a good thing.


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Realtime PCR plate cleaning
Have anyone used realtime PCR, is that the plate for realtime PCR is one-time using or not ? Otherwise how to clean the plate after reaction to prevent wrong result in next time using ?

ahh... it is usually used only for one round.. but if your experiment is not very sensitive then you can wash it in hot water and autoclave it. (I think they are autoclavable)

@WYSIWYG thank you so much, can you describe more what matter occur to DNA when they being autoclave ? Are they denatured ?

nothing happens to the DNA.. it might get damaged but we cant rely on that. autoclaving is just for making it sterile. hot water wash should remove the DNA.. you may use a mild soap/detergent too but make sure that none remains.

Ok, thank you so much, i hope it will work. Otherwise i will try another way to reuse the tube.

A possible solution is to use the DNA ZAP solution, which I know many people used in adjacent labs and were happy with for cleaning their DNA free workbench. The product sheet states

When 500 ng of template DNA is dried down in a PCR tube, it is rendered unamplifiable upon treatment with DNA ZAP solutions. Additional experiments demonstrate that the DNA is degraded down to free nucleotides.

Please note that I'm not promoting any specific product here and you can purchase any similar product/technology for your particular application. Obviously the best recommendation is to use new plates for each reaction but this/or a similar product can be an option too although the risk of DNA contamination in a PCR based reaction should be carefully balanced against simply using fresh plates considering the product itself is not free either.
The product sheet provides instruction for cleaning/treating PCR tubes hence I assume you use the same protocol for plates if you have the plate centrifuges available in your lab. You can get rid of the solutions by tapping the plates on a tissue to get the liquid out of the wells.

Honestly spoken I would never think about re-using the PCR plates. Sure, they are not for free, but the reagents of the assay aren't either. If you blow up one experiment because of a contamination (and you will likely use the same genes in an assay more than once), this is more expensive (plate, reagents, sample preparation) than what you save. Besides that, these commercial solutions aren't free either.

@Chris I 100% agree! I would personally never re-use anything for any reaction and its just best to use new consumables but just though I provide a possible solution to the OP question, which might also be of interest to others but again I agree with you and I would personally never re-use any consumables specially for reactions such as PCR!

@Chris I changed the language used in the response to highlight it as a possible solution and not a suggestion and clarified that the best thing to do is to use a new plate.

One of the claimed virtues of RT-PCR plates is their inability to bind DNA. (See for example the assortment at http://www.fisher.co.uk/product/brand_listing.php/T/Thermo%20Scientific%20ABgene/PCR%20plate ). As such, all you need to do to remove the DNA (genomic, amplified, or primers) is to rinse them. This suggests that rinsing may also work for dNTP.
On the other, many types of RT-PCR plates are made of polycarbonate, which is able to bind protein. I would be concerned about an uneven amount of polymerase sticking to the well. To go around that, you should re-use, in any PCR plate, only those wells that saw polymerase before.
I have no idea whether other molecules bind to the wells, but presumably they fall on the DNA or protein paradigm.
It will cost you only one plates and regents for as little as four reactions to test whether these plates are reusable. In the first run, measure two samples, A and B, for some transcript. Rinse the wells, and measure samples A and B again. If the transcript ratio A/B is the wildly different between first and second attempt, you know you can't reuse them. I would also be concerned if it will take many more cycles to reach the plateau stage at the second attempt. Passing these tests, you may be all set. Failing these tests, you may still be able to use the remaining wells. Let us know.

Welcome to Biology SE! Can you clarify what you are asking?


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NAG, FUC molecules in PDB files
In some proteins (such as 4ZXB, 6CE9 which are respectively apo and halo forms of insulin receptors), I see ligands such as FUC (ALPHA-L-FUCOSE) and NAG(N-Acetylglucosamine). No matter which paper I checked I see no explanation for these ligands and therefore I assume they have to do with the experimental procedure but I do not quite understand what is their role. Do they stabilize a particular state of the protein? Note that the proteins above are determined by different methods the first one X-ray crystallography and the second by electron microscopy. I have no technical knowledge about the neither method so any input or link to a paper that actually explains them would be appreciated.
Thanks

The insulin receptor is a glycoprotein. This is discussed, albeit briefly, in both papers for the structures you mentioned and the glycosylation is visible in the structures themselves. You can get more info in the following paper, among others:
Sparrow LG, Lawrence MC, Gorman JJ, Strike PM, Robinson CP, McKern NM, Ward CW. 2008. N-linked glycans of the human insulin receptor and their distribution over the crystal structure. Proteins 71(1):426-439.

Yes this is the right answer. However I would make the following point (which you are welcome to incorporate). The PDB entry page visualizes manose, fucose and N-acetyl glucosamine as individual sugars. The paper you cite involves painstaking chemical analysis to show how the sugars are linked together into glycans (GlcNAc2Man6, GlcNAc4Man3Gal2Fuc etc.) and where they are linked to the protein. This emphases that the interpretation of X-ray crystal structures assumes linear polypeptide chains, but has no basis for assumptions on sugars, and the resolution is insufficient for this.

Thanks, since I was particularly interested in the insulin protein I will select this as the correct answer. As a side note to anyone who is interested this article discusses possible functional role of glycosylation for insulin: 1- https://www.sciencedirect.com/science/article/pii/S0167488900001099
2-http://www.jbc.org/content/274/32/22813.full

Apologia
This addresses the general case, rather than the particular case of the insulin receptor, which is answered by @canadianer.
The General Problem of unexpected ligands in PDB structures
In preparing a relation database of about 400 protein structures I encountered the problem of distinguishing ligands that were substrates or cofactors of the enzyme from those that were not. And, believe me, the latter were widespread (and a real pain). In some cases it was obvious that the ‘ligand’ was the buffer (often MES) in which the protein was dissolved or ions such as sulphate. In the case of sugars, I assumed that the latter were present to aid crystalization. That view seems to be supported — in some cases at least — by a review by McPherson and Gavira. In a list of eight categories of additives that are used in protein crystallization they include:

(v) Osmolytes, co-solvents and cosmotropes are compounds that exert their effects at relatively high concentrations, 1 M or more, and include a wide range of molecules such as sucrose, trehalose and other sugars, proline, TMAO, glycine, betaine, taurine, sarcosine and a host of others. The effect of their inclusion in the mother liquor is to stabilize (or destabilize) the native conformation of the protein by altering the interaction of the surface of the protein with water, or by altering the hydration layer and possibly the structured waters.

Example
As an example I cite the seven molecules of glycerol in 1B6G (shown below), a haloalkane dehalogenase from the bacterium, Xanthobacter autotrophicus. Although certain, usually pathogenic, bacteria do have glycosylation pathways, there is no suggestion that this is the case here, as indicated by the nature of the triose and the fact that the crystallization was performed in a glycerol solution:

Subsequently, the crystal was equilibrated for 0.5 h in a solution containing 70% ammonium sulfate and 100 mM MES buffer pH 5.0 and was then soaked for 3 h at room tempera- ture in 10 mM 1-chloropentane, 70% ammonium sulfate and 100 mM citrate buffer pH 5.0. A solution of 30%(w/v) PEG 6000, 20%(v/v) glycerol and 100 mM citrate pH 5.0 was applied as cryoprotectant during data collection.

Haloalkane dehalogenase, 1B6G. The glycerol molecules (grey and red) are shown in space-filling mode. (The yellow and red molecule is sulphate, the green one a chloride ion.)


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Statistical Coupling Analysis (SCA) to identify coevolved residues: use of ICA
We've begun to try out the SCA Matlab toolbox (latest version) downloaded from Dr. Rama Ranganathan's website, and, following the included tutorials, would like to apply it to our protein family. The SCA calculation involves determining independent components (IC) of positional covariation by applying the "Infomax" algorithm to a few top eigenmodes (that have been obtained from the positional correlation matrix). Here, we observed that the output (IC vector weights) seems to depend on the no. of top eigenmodes chosen as input (i.e. on choice of the parameter kmax). If this is indeed the case, then is there some "working rule" to optimally choose kmax (say, based on the number of significant eigenmodes turning up)? Since this isn't made very clear in the tutorials (at least to me), I thought of trying my luck here.
Would really appreciate some guidance/pointers from SCA users. Thanks a lot!

This question is has a very narrow scope. Please post this in its designated google-group or other forum.


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What's the difference between growing cells in culture and cloning them?
The wikipedia page on Hela cells refers to George Gey being able "to isolate one specific cell, multiply it, and start a cell line." Later it says, "In 1955 HeLa cells were the first human cells successfully cloned."
What's the difference between multiplying the cells and cloning them? How is multiplying not cloning?

Clones are genetically identical daughter cells from one parent cell. So when you start you culture from one cell, all new cells are clones of this cell.
The first three cells (red, green and blue) undergo clonal expansion.

If you multiply cells in cell culture you usually start with thousands of cells, as this can be seen in the second part of the figure. These cells are not genetically identical, as they individually collect mutations. So the daughter cells of each "starter cell" (a lot of them) in the mixture are technically clones, but the whole culture is not seen as clones. See also the Wikipedia on this topic.

Ok, makes sense. So when the Wikipedia article refers to "isolating one specific cell" and multiplying it, is that just cloning?

Yes, that would be cloning. If you make a very high dilution of a cell suspension so that you can grow cells on a solidified growth substrate, then every daughter cells which derives from a single cell is a clone.


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estradiol hormone role
Increasing estradiol (E2) hormone from ovarian follicles prior to ovulation has been hypothesized to play a critical role for induction of pheromones. These pheromones render females sexually receptive to males to facilitate mating. An investigator performs experiments in sheep in which females are gonadectomized, then treated with E2 or vehicle alone and allowed to breed. What can be the hypothesis

The experiment is easy to design: Measure the concentration of the pheromones in the test sheep versus the control sheep and see what the outcome is. Further compare the reaction of the male sheep on both, control and treated sheep. Ideally you treat both animals with some kind of treatment to avoid an influence by the application method of the estradiol. Also blind the researchers to avoid their influence on the experiment.
The hypothesis would be: Has estrogen E2 an influence on the pheromones in female sheep and do these pheromones influence the male sheep.


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How do the first and second law of thermodynamics adhere to breakdown of glucose?
I know the first law is energy can only be converted from one form to the other, therefore in the breakdown of glucose 40% potential energy is converted to ATP and 60% is converted to thermal and unusable.
Does the second law adhere to the breakdown of glucose? Isn't it just the same as the first?

I'm not sure what you're asking.  The first law states that total energy in a closed system is constant, whereas the second law states that systems tend toward an increase in entropy.  I don't understand how your question relates to these topics, but the laws of thermodynamics have yet to be violated, even in biology.

Second law adheres to all spontaneous natural processes, including all biochemical transformations occurring spontaneously.

The Second Law of Thermodynamics is intimately linked to the thermodynamics of the breakdown of glucose. By "the thermodynamics of the breakdown of glucose," I mean: is the breakdown of glucose spontaneous, is it thermodynamically favorable?
You may have seen the criteria for the thermodynamic favorability of a reaction in terms of Gibbs Free Energy:
$$\Delta G = \Delta H - T\Delta S\leq0$$
That is, for a reaction to be favorable, the change in Gibbs free energy from the initial state (glucose) to the final state (carbon dioxide, water, and energy) must be negative. This equation is valid for systems at constant temperature and pressure, which is assumed to be satisfied in biological systems. According to Wikipedia, $\Delta G$ for the breakdown of glucose is negative, $\Delta G = -2880$ kJ/mol.
However, there is a nice derivation on Wikipedia that the above equation is nothing more than a statement of the second law of thermodynamics, applied to the case of constant temperature and pressure. So in sum, the second law of thermodynamics is the reason that glucose can be broken down in such a way that useful energy is released in the form of ATP, i.e. that the reaction is spontaneous or thermodynamically favorable. Saying that the Gibbs Free Energy of the breakdown products is lower than that of glucose itself is the same as saying that the breakdown of glucose does not decrease the total entropy of the reaction and its environment.


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genomatix software linux install
I have installed the genomatix software which is a jar file the name is InstallChipInspector21.jar.But now I really don;t know how do I start it ,can any one suggest me a method??

This question appears to be off-topic because it is about a computer package installation. Refer to the readme associated with the package for details on this or contact the developer.

well it's not about installation I have installed it I need to run the program,that is what I'm asking for

I don't know what else to say as an answer but here is a link to the user manual. If you are looking for more specific usage instructions, I would suggest that you try Biostars though I can't guarantee that you receive an answer.

I have seen the pdf it doesn't talk about how to start the program...

@krushna Try the command java -jar InstallChipInspector21.jar ?

yeah I have installed the software using the command but I need to run it how do I do that??


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PAH gene mutation
Analysis of the DNA of the Phenylalanine hydroxylase (PAH) gene in a
patient with phenylketonuria revealed a mutation in the protein coding
region whose predicted effect would be to replace the amino acid aspartic
acid, with histidine. Nevertheless, no mutant protein could be found in the
patient’s cells.How this could be as mutation is in the protein coding region,can anyone explain?

Can the mutation be in an intron? Otherwise I assume that some kind of post-translation modification is involved.

Can you be a bit more specific about the mutation?

Please add more details.. When you mean DNA is mutated do you mean one allele or both..

In terms of the way that the question is worded, I imagine the most likely answer would be that the amino acid substitution D > H disrupts the folding of the protein so that it is recognised as being aberrant and is rapidly degraded.

There are many possible reasons for this, the two most obvious ones are:

Alternative splicing. Perhaps the mutation is indeed in the coding region but of an exon that is not constitutively expressed. As a result, the majority of transcripts created from this gene would not contain the mutation and neither would their resulting protein.
Post translational modifications. Proteins with the mutation might actually be produced but the histidine is changed to an aspartic acid by post translational modification so the mutation is not detectable in the cell's proteins.


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Are scientist able to correct mutiple gene defect in our body by using CRISPR
Are scientist able to correct mutiple gene defect in whole body by using CRISPR recently?
AS i know, it is in a beginning stage

Certainly not yet - there is not yet any evidence that this can be used to correct specific deficiencies in an adult, though the in vivo applications are growing.
In particular, an exciting recent advance is the opportunity to better understand carcinogenesis by 'editing in' certain known mutations into a mouse, say, that should cause a cancer, and watching for when the cancer actually arises...


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Genome Assembly Using Reads
I'm taking an online bioinformatics class and I am stuck on a problem. The resources I have found don't help that much. Here is the problem:
"Assemble the error-free reads from a circular genome on the dataset below. You do not need to consider the reverse complement of the reads.
Dataset: CGTTCT CTAG GACGT TAGAC
As the reads do not have the same length, convert the reads to 4-mers using the approach proposed by Idury and Waterman."
I believe this means I need to convert this into sets of 4 characters each. Do I simply break them up by taking the first 4 characters and last 4 characters like this?
CGTTCT: CGTT --> TTCT
CTAG: CTAG --> CTAG
GACGT: GACG --> ACGT
TAGAC: TAGA --> AGAC
And end up with a new dataset of: "CGTT TTCT CTAG GACG ACGT TAGA AGAC"?
Or is there more to it than that? Thanks for any help.

I would guess that it would be all 4mers. In this case that would mean that CGTTCT would also yield GTTC. But as I say - this is a guess.

It does in fact mean all possible 4mers. I don't know how much material you get from the course, but they refer to this paper: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.79.6459&rep=rep1&type=pdf

To be sure I'm understanding correctly, does that mean the new dataset should be: "CGTT GTTC TTCT CTAG GACG ACGT TAGA AGAC"?

Actually the new data set will be,
for k = 4 (k-mer value)
CGTTCT -> CGTT GTTC TTCT
or

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874646/


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Drosophila Crosses
I have 2 transgenic lines of Drosophila. I would like to cross them and get both transgene in the same fly. To start this cross, does it matter which fly is female or male? Or as long as the female is virgin, it does not matter?

flies aren't my field of expertise, but you probably would want to verify by PCR that any crosses were homozygous in any case.

Ah no, I have certain markers for it. So thats not a big problem. I am asking a really silly question probably.

As long as none of your genes are X-linked and you use virgins it shouldn't matter and you'd get the same proportion of double homozygotes. You may want to perform a reciprocal cross (https://en.wikipedia.org/wiki/Reciprocal_cross) if you don't know what chromosome these genes are on (though flybase.org is useful too).


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Do RNA molecules bind each other?
I have a question, maybe a naive one.
Let's assume that we isolated some RNA from a tissue. Do RNA molecules can bind each other if they have the complementary sequence? I know that some small RNA molecules can bind other RNAs and regulate them but I do not know it is possible in this case? It is like 2 different mRNA can bind each other or not?

The difference between RNA and DNA is rather small, and both can form a double-helix structure. So if you had two sequences of RNA complementary to each other they would basepair and form a helix.
There were also some ideas to use this for therapeutic purposes, antisense RNA, an RNA oligo complementary to a messenger RNA, can theoretically be used to silence gene expression by base-pairing to the mRNA.

Thanks. But do they need to be completely complementary or not? And during the experiments, is there a way to prevent this issue?

@golgicik Not completely, a certain amount of mismatches would be tolerated. There are various programs to calculate the likely secondary structure of RNA like MFold or the Vienna RNA package you could use to predict the structure of your specific RNA.

Thanks @Mad Scientist. I think 2-ME would prevent it, right?

@golgicik 2'-OMe doesn't really affect base-pairing, it's a modification of the ribose.

I mean 2-Mercaptoethanol.

Would ME prevent secondary structure? I think at reasonable concentrations it would have little effect and it's not used (as far as I know) as a denaturant.

@golgicik 2-ME doesn't denature secondary structures. It is a reducing agent that reduces disulphide bonds in proteins. Urea will denature RNAs just like it does  proteins.

@golgicik for hybridizing two RNA strands you can use UNAfold. However, it doesn't compute it for RNAs longer than 500nt. Also, in RNA G can form a wobble base pair with U

@MadScientist answer is very good. I just want to add a detail that could not fit in a comment.
Double stranded RNA is nothing exceptional. You can see an RNA strand that binds to its antisense in tRNA and in RNAi for example.
tRNA

RNAi


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Elution of biotinylated RNA
I am using Neutravidin Agarose beads to isolate biotinylated RNA. The thing is, in the manual it says to use 8M Guanidine-HCl, pH 1.5 for elution biotinylated molecules. As far as I know it is a very harsh elution buffer. Do you think it would effect my RNA? I have isolated my RNA with TRIzol which also contains Guanidine salt but the concentration is not that high.
As a second question, do you know other ways to elute my biotinylated RNA from beads?
Thanks!

The biotin-streptavidin interaction is extremely strong, one of the strongest (if not the strongest) non-covalent interaction between biomolecules. So you'll need pretty harsh conditions in any case.
Extreme pH values are generally a very bad idea for RNA. I'm pretty sure that the conditions you mentioned are not meant for elution of nucleic acids. Such low pH values (and high pH values as well) lead to a quick hydrolysis of the phosphoester bond of RNA, you can read more about the mechanism of that in this review.
A quick Google search lead me to the following protocol:

To dissociate biotinylated nucleic acids from Streptavidin-Coupled
  Dynabeads®, incubate the beads in 95% formamide + 10mM EDTA, pH 8.2
  for 5 minutes at 65°C or for 2 minutes at 90°C.

This sounds reasonable to me, as formamide is also commonly used in sample buffer for denaturing gel electrophoresis of RNA. I haven't done anything like that myself, so I can't guarantee that this would work in your case.

Hey,
thanks a lot for your answer.
I found an other solution. Instead of destroy the bond between biotin and Neutravidin which is obviously so strong, I am going to cleave the bond between biotin and RNA which seems more logical for downstream experiments. The biotin that I am using is binding to RNA with disulfide bonds. So, I am going to use 100 mM 2-mercaptoethanol to achieve it. What do you think about it?

@golgicik Cleaving at a different point is the easiest way if you don't need the biotin later and only use it for purification. I can't comment on the practical aspect as I haven't done something like that myself.

I will use RNA in RT-qPCR so I do not need biotin. Thanks a lot for your input!

i guess pH 8 won't be good for RNA. Mercaptoethanol or DTT would be a decent option.. I am not sure about this but you may use proteinase-K as well.. It would destroy streptavidin and other protein contaminants.

I'm way late to comment, but thought I should add that I was able to displace biotinylated protein from avidin with a large excess of free biotin. I can't remember the exact concentration, but it was pretty high, and I had to let it sit overnight.


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Why do we suffer abdominal pain after we get very angry or stress?
You also realize probably, when we get so much anger or high levels of stress, after some point we have terrible stomach ache, like really heavy pain. Why is that? How stress or anger can effect our stomach? What is the mechanism?

Cortisol produced from the zona fasciculata of the adrenal cortex is directly caused by stress.
According to http://en.wikipedia.org/wiki/Cortisol

Cortisol stimulates gastric-acid secretion. Cortisol's only direct effect on the hydrogen ion excretion of the kidneys is to stimulate the excretion of ammonium ions by deactivating the renal glutaminase enzyme. Net chloride secretion in the intestines is inversely decreased by cortisol in vitro.

This increase in gastric-acid secretion can irritate the lining of the GI tract, especially if there are ulcers or inflammation, resulting in pain. In addition, the fight-or-flight response causes

inhibition of stomach and upper-intestinal action to the point where digestion slows down or stops.

(see http://en.wikipedia.org/wiki/Fight-or-flight_response)
Those things, paired with increased muscle tension and vasoconstriction cause pressure changes in the GI tract, which can cause pain.


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A question related to qPCR analysis
Here is the thing. I am using a method, called TU-Tagging, to isolate cell type specific RNA. You can find more about the method here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783170/
To explain it very briefly, there is an enzyme called UPRT (Uracil Phosphoribosyltransferase) which is expressed in Toxoplasma gondii. Under normal circumstances UPRT creates UMP from host cells' uracil and this UMP incorporated in RNA. However, they found that this enzyme cannot distinguish uracil and uracil analog 4-Thiouracil (Basically has thio on 4th position of the uracil). So, when 4-TU provided externally, UPRT incorporates 4-TU into RNA and you get thio-RNA. Then with basic biotinylation and streptaividin isolation you can get thio-RNA from your total RNA isolate. (Of course UPRT should be put to specific cell types of your interest)
Anyway, after the explanation (maybe it was unnecessary though) here is my question.
After I isolate my so called specific RNA, I check the purity by qPCR by using cell type specific markers. I do not have a reference gene. The only thing I am interested in is the presence or absence of markers (and also the fold change of course). Now, I have some data but I do not know how to present them (There is nobody in my lab experienced with qPCR and my experience is just so limited).
Do you have an idea how I can overcome this issue? I looked into some calculations but most of them are needed for reference genes or some other stuff. The only thing that I have is cycle numbers and my primers' efficiency. Do you think I can do something with these inputs? (I know that cycle numbers mean nothing in a plot, so I do not want to put cycle numbers basically.)
Thank you for reading it. Any help/idea would be very appreciated. I am so desperate. Thank you.

Great/clear/easy to follow explanation of the TU-tagging technique. However, I am confused as to what you are really asking for. You isolated a cell specific RNA and you want to check the "purity" of your isolate? What do you mean by this?

Thanks. So imagine like, I have a fly line which has the UPRT in their neurons. When I do the TU-Tagging experiment, in theory I just isolate RNA from neurons. So, I should not see any glia specific markers lets say. So, with qPCR I am checking cell type specific markers in order to see my RNA is clean or contaminated by other cells.
However, I do not know how to plot these qPCR results. I just have cycle numbers and do not know how to present them.

Does your qPCR software not provide you with the progressive graphs where you can see each sample crossing the threshold? That might be a nice picture to show.

OK, let us start from the beginning. We know what makes one cell type different from another cell type is its expression - i.e. what genes are actually being transcribed into RNA. Therefore, there should be certain RNA in one cell type that should not exit in another cell type. If you isolate RNA from a certain cell type, say a neuron and you want to show that your sample RNA contains only neuronal RNA, than you have to prove 2 things:

That your sample contains certain RNA sequences that are known to be in neurons
That your sample does NOT contain certain RNA sequences that are known NOT to be in neurons

There are several lab procedures for identifying the presence/absence of certain RNA sequences in a sample, your chosen technique is qPCR. Here is a brief explanation of how qPCR works.
You put your RNA sample along with a sequence specific primer in the qPCR machine. The primer will bind to a specific sequence and then that strand will be replicated. This will keep on repeating, cycle after cycle. So if you start with 1 sequence of say sequenceA, after cycle 1, you will end up with 2, then 4, then 8 and so on.
Anyways, if your goal is to use qPCR to prove your RNA sample comes from a neuron then you must find some sequences that are known to be specific to neurons. Then find primers for these sequences. Then use these primers to amplify some RNA from your sample. If you get amplification, then you must have had that sequence in your RNA sample. To further support yourself, you may also want to look up a few sequences that are known NOT to be expressed in neurons and using the same principle prove that these sequences are NOT in your sample.
The above will give you qualitative data, it will simply tell you if a certain sequence exits or does not exist in your RNA sample. Since you are only interested in the presence or absence of markers, this should be sufficient. Howver, if you would like to quantify your data, i.e. find out how much a certain sequence is expressed then read on.
You can also get quantitative data from qPCR (after all qPCR stands for quantitative PCR). The number of copies of an RNA sequence after a certain number of cycles is proportional to the initial number of copies of that sequence in your sample. In other words, if your initial RNA sample has a lot of sequenceA and a little sequenceB, then at any cycle, you should see more sequenceA.
We can take advantage of this relationship. There are two general methods you can use to quantify your results. One is called Absolute quantification and the other Relative quantification.
The idea behind absolute quantification is that you run two qPCRs. In one, you do your RNA sample. In the second, you run a standard sample, for which a standard curve is available. You can then compare your RNA sample results with the standard's results to get an idea of the number of copies in your sample.
The idea behind relative quantification is that you compare the qPCR results for your sequence of interest with the qPCR results of a refrence gene. This will only give you an idea of how many times more or less your sequence of interest if expressed than the reference gene. Since you do not have a refrence gene you cannot use this method, but you can use the absolute quantification method explained in the paragraph above!
Here are very useful links on quantifying using qPCR:
http://en.wikipedia.org/wiki/Real-time_polymerase_chain_reaction#Quantification_of_gene_expression
http://relative.gene-quantification.info/
Let me know if you need further clarifications.

Hey thanks! As you told also I am just interested in to show presence and absence. I already did lots of qPCR experiments with neuronal and glial primers and have the data as cycle number. So I just want to show them but I can not show the cycle number, I need to express it as some numbers which will show fold change for example. I need to plot the data. So can I just assume that every cycle will double my cDNA and could use an equation like this: 2^-Ct ? Or it is not the way?

if "C" is your initial concentration, and "t" is the number of cycles, then yes your equation will tell you the fold change. Sorry for the late response, haven't been on here for a while.


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In vitro transcription, contamination problem
I am using a RNA which is in vitro transcripted before I started my project. It turned out it is not prepared properly and has DNA contamination.
Instead of perform the in vitro transcription again, I want to try to clean it with DNase treatment and LiCl precipitation afterwards. Do you think would that work?
And second thing, in vitro transcipted RNA is in different dilutions, so I have a little bit of stock (maybe 5 uL) which is around 200 ng/uL and I have other dilutions 1 and 0.1 ng/uL. I would like to clean all of them. Do you think if I just mix all of the dilutions with the stock and determine the concentration and do the rest? Is it appropriate?
Thanks!

DNase treatment is actually one of the optional steps if you want to get rid of the original DNA template: https://www.neb.com/protocols/2013/04/02/standard-rna-synthesis-e2050
If you know, your the contamination happened after the transcription, DNase treatment should obviously help.
Concerning the aliquots: your stock is 200 times more concentrated than the aliquots (so to get equal amounts of RNA as in 1 µl of the stock, you'd need to take 200 µl of the 1 ng/µl aliquot). I don't think it's worth mixing them (and the efficiency of RNA precipitation will likely be decreased), otherwise just make another transcription.

Thanks @har-wradim. You are right about the aliquots, I will just try to clean the stock even though it is not that much.
I think the contamination is due to original DNA template. Do you think it would still help?

It should, that's precisely the purpose of the optional DNase treatment step. For many tasks the presence of the DNA template is not a problem though.

I am mixing it with another RNA and performing qPCR. So for me, I need to see if I get the exact amount of RNA that I put after certain steps. And with -RT reaction of this RNA (without RT enzyme) it gives signal. That's why I am not sure if the signal that I get from my experiment belongs to my RNA input or DNA template contamination.


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How to convert nanogram to picomole?
I have lets say 10ng/uL of RNA. How can I convert it to picomole?
I need it to calculate how much magnetic beads I need. This is the product: http://tools.lifetechnologies.com/content/sfs/manuals/dynabeads_m270_sav_man.pdf
I do not know how much beads should I use.

Do you want the the concentration or the pc/ul? Also what is the mass of your RNA?

To make the calculation, you need the mass of the DNA. What would also work is the sequence from which you can calculate (or estimate, depending on how exact you need this) the mass.

The thing is I have no idea about the mass. It is a pool of transcripts from a specific cell (they are biotinylated) and I need to pull them out with magnetic beads.

Average molecular weight (MW) of a ribonucleotide = 340g/mol
Size of RNA = x
MW of RNA = 340*x g/mol
moles of RNA needed = y * 10⁻¹² moles (1 picomole = 10⁻¹² mol)
mass of RNA needed = 340*x*y*10⁻¹² g
Conc of RNA = 10*10⁻⁹ g/µl `(1ng =10⁻⁹g)
Volume needed for y picomoles = (340*x*y*10⁻¹²)/10*10⁻⁹ = 0.034*x*y µl
If your RNA is biotinylated then add the MW of biotin to MW of RNA

I don't think it would be appropriate to take account of the biotin, since the RNA concentration (ng/ul) has presumably been estimated by a method that only measures RNA.

yeah thats true.. Plus with a big RNA the biotin tag won't matter..

Thanks for all the answers. The thing is RNA is containing biotin for every uracil that it has.(TU-Tagging method: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783170/) I decided to use an estimated amount of beads since I do not know the amount of biotinylated RNA in my sample.


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RNase and EtOH issue
I work with RNA and I have some RNase issues. For example I am using RNeasy mini kit and at one step I use 100% EtOH. It does not say it is RNase free or not. Does absolute EtOH inhibits or removes RNase or should I be worried about it?
Addition: I am using eppendorfs directly from the bag. So they supposed to be RNase-free, right?
Thanks.

If nobody else used your EtOH it should be RNAse free, the same is true for tubes. I used tubes fresh from a new bag without problems. But again: Make sure they are handled with gloves all the time and that nobody else uses them.

I have my own bag for tubes and I am taking them from the bag and putting in a box and I use that box only in "RNase free area". Would that be OK?
EtOH is an 1L bottle and everybody taking aliquots from it actually. I have also my own and just me using it.

Ethanol is not a problem, we use it without any further precautions and it seems to be RNAse free. You should take the usual precautions you should take with all chemicals when you work with RNA, never use a spatula or spoon to get anything out of the bottle (unless you want to bake your spatulas all the time), always pour them to avoid any contamination.
We autoclave the Eppis once, this might be an unnecessary precaution. I think you can also buy them certified RNAse free, but I'm not sure if they are actually treated any different or simply more expensive.

Thanks. What do you think about glass bottles? For instance, I am preparing RNase-free buffers and putting them clean and autoclaved bottles. Do you think it is ok or should I just use plastic containers?

I'd probably clean the glass bottles with DEPC first to get rid of any RNAses. Usually baking at 200-250 °C is the way to go for glass, but the bottles typically have some plastic which would not survive this. I also tend to just reuse the same bottles for identical content, then you don't have to make them RNAse-free again.


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RNA isolation from Drosophila head
I need to isolate RNA from Drosophila head. I basically chop the head off and first homogenize it with a homogenizer (similar to this: http://www.omni-inc.com/omni-tissue-homogenizer-th-package-p-11.html) and further homogenize it with 22G needle in TRIzol reagent. I use TRIzol RNA isolation for larvae brain a lot and have no problem with the method itself. However, with adult I got really low RNA concentration which is weird.
I thought that maybe I have problem with homogenization itself, or I overlook something (such as extra centrifugation or different kind of homogenization etc). Does anybody have experience with it?
Thanks.

You mean you have not a lot of RNA compared to whole larvae? Don't forget that in larvae a lot of gene expression is going on compared to the brains of developed flies. And the heads are a lot smaller, too. What does low concentration in terms of numbers mean?

I isolate RNA from larvae brain so not whole larvae. For example, from approx. 150 larvae after DNase treatment, I got more or less 120 ug RNA.
For the very first time I have tried with adult. I used 220 adult heads and after DNase treatment concentraion of RNA was 52 ug. For me this result indicates a problem.

Oh, then I misunderstood that. How is the quality of the RNA?

I measured with nanodrop and these are the ratios for adult RNA: 260/280= 2.17 and 260/230: 2.21
For larvae I usually get also more less these ratios.

That sounds not bad for me, at least you have no quality problem. Do the heads dissolve completely?

Good way to get heads off (if doing many flies of the same genotype/treatment etc.) is to put whole flies in a safe-lock eppendorf, put them in liquid nitrogen, drop the eppendorf on the desk a few times and the heads should have fallen off and you can collect them - ideal if you are putting them in liquid nitrogen anyway (as is common with RNA extraction).

Yes quality is ok but I need more RNA, thats the issue unfortunately. I also homogenize larvae brain with 22G needle and works well. This time after homogenizer, I did with 22G needle also, it was going up and down easily. After phase separation with chloroform, there was a huge trash phase.
I did not do any liquid nitrogen step before homogenization. Do you think it can be the problem? I simply make them sleep on ice and then got their heads.

You say that 150 larval brains give you ~120 ug RNA. Do you have an idea of the amount of total RNA that can be extracted from a single brain?
I have posted a question here:
http://biology.stackexchange.com/questions/28078/how-much-total-rna-can-be-extracted-from-drosophila-brain

It doesn't seem like a homogenization problem. Nonetheless you can try this:

Suspend the heads in trizol
Homogenize by passing through 30/31G needles (insulin syringe) up to 10-15 times.
Heating a little might help but I think it is unnecessary for these tissues.

Insulin syringe is much better than the hand held homogenizer. Freeze-thaw 1-2 times if you want (helps in homogenization).
During extraction you can add 5M LiCl (100µl) + Ethanol (1ml) to 400µl aqueous phase (instead of isopropanol), keep at -20⁰C for 1-2h before centrifuging. Alternatively you can add a little RNAse free glycogen to Isopropanol during precipitation.
PS: the liquid Nitrogen trick that rg255 mentioned is for quickly getting heads and avoiding the pain of dissecting each head.


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RNA integrity and RNA fragmentation
After RNA isolation and before following experiments, I know that RNA quality so called RNA integrity is so important. So why there is a RNA fragmentation kit? Do we need RNA intact or fragmented? (RNA will be used for biotinylation and RT-qPCR afterwards)

This paper uses a similar technique to you by purifying total RNA, synthesis of double-stranded cDNA containing a T7 promoter sequence, cDNA purification, in vitro transcription in the presence of biotin-labeled ribonucleotides and this transcription is possible with T7 RNA polymerase since the cDNA contains the T7 promoter region, and finally fractionation of RNA. This then allows target biotin-labeled RNA for short (~ 25 nucleotides) oligonucleotide arrays. The T7 polymerase performs a linear amplification, and the in vitro RNA obtained reflects the abundance of each transcript in the initial RNA.
Hence I assume you follow a similar protocol although I'm not sure why you do RT-qPCR on the fragmented RNA since that is unlikely to work if you are doing it using primers for specific regions as everything is fragmented. So I think you do a RT-qPCR before the fragmentation to test the expression levels of certain (known) genes (to see if you are getting the expected results) before you go ahead with fragmentation and look at the global RNA expression. If my assumptions are wrong, then it would be great if you added further details of your experimental protocols with the different steps and the exact orders of the steps followed and the results assessed and the experiments performed so that I can modify my response.