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Learn more about Cars Micro Drifters Multi Launcher:
Manufacturer: Mattel 1001134
Mfg Part#: 737EA368
Sku: 258414425
Package Weight: 1.23 lbs
Batteries: Not Applicable
See more in Die-cast
|TRADE QUOTE SHEET COPY ONLY. NAMES AND PRODUCT DETAILS SUBJECT TO CHANGE. PENDING LICENSOR APPROVAL. BCD20 MICRO DRIFTERS Multi Launcher Featuring DisneyoPixar's CARS: Amp up the animated action with this launcher that can propel up to three Micro Drifters vehicles at once! Each character completes a loop before being fired out to start the race at the World Grand Prix. Shoot the vehicles out and watch them race, drift and swarm - just like they do in the films. Includes 1 Micro Drifters vehicle and 1 launcher.


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Originally written on Sportress of Blogitude | Last updated 1/10/13
On Wednesday, regular followers of the official Twitter account of the Boston Red Sox were likely taken aback a bit if they noticed that the account's avatar was not the traditional BoSox logo but instead the image of some wildman with unkempt hair and a ratty goatee. But any fears of some hostile takeover by some unstable, weirdo Red Sox fan were unwarranted and soon allayed, as it was quickly discovered that the man whose image became the Red Sox's Twitter account avatar was none other than the team's new closer, Joel Hanrahan. And given the few tweets he did offer up on Wednesday, he didn't do half bad with his assignment. Well done, crazy-haired closer guy. [BuzzFeed] TweetThe post Some wild-haired crazy man hijacked the Red Sox's official Twitter account (pic) appeared first on Sportress of Blogitude.
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subset: commoncrawl


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Available for sale is our beautiful Sunbeam Tiger MK1A. Black with Red interior. Genuine 2 Owner car. Totally rust free. Restored/refurbished to exact factory... More
1965 Sunbeam Tiger Mk I GT RoadsterTo be OFFERED AT AUCTION at Auctions America's California event, July 17-18, 2015. Click here to view this... More
- Listed in the Tiger registry- Upgraded 347/500 HP engine- Aluminum heads- Dyno sheet- Transmission built to match engine by ATI- 9 Inch Ford... More
- 289 V-8 engine- 4-speed transmission- Unrestored- British Racing Green with Black interior- Authenticated by STA- LAT1 super induction kit- LAT8 Tiger valve covers-... More
- Comprehensive rotisserie restoration- 302 CI high performance engine- Tremec TKO 5-speed transmission- Dale's Restorations complete front end- Power assist steering- Wilwood 4-wheel disc... More
1967 Sunbeam Alpine Series V. Same owner for past 10 years, straight body, no rust. Vehicle is black, with soft top. Engine is 1725cc... More
Sunbeam Tiger 1965 V8 260 rare car to find, projectcar. Original 1965 Sunbeam Tiger. This Sunbeam Tiger is the V8 version of the Sunbeam Alpine... More
VERY NICE SUNBEAM TIGER MARK 1-A WITH HARD TOP. NICELY RESTORED. SILVER WITH BLACK, THE SUNBEAM TIGER IS AN ENGLISH BUILT SPORTS CAR WITH... More
1967 Sunbeam Tiger MKII. 1 of 530 made. V8 289 Ford 4 speed. Dual quad 331 stroker steel crank. 400 easy horse power. Lots... More
1966 Sunbeam Tiger MK1A If you are interested in this vehicle or have any questions, please call Maurice at 250-724-4672 This car started life as a SBF... More
With an illustrious history, beginning in 1888, Sunbeam started out manufacturing bicycles but soon transitioned into motorcycle manufacturing and then automobiles in 1899. During... More
Sold new by a local northeast Pennsylvania dealer who, upon taking it back in trade, decided to keep it for himself. It is a numbers... More
1967 Sunbeam Alpine V1967 Sunbeam Alpine V model in mint conditionAston Martin Almond-Green metallic exteriorEquipped with a 1725ccm 4-Cylinder and a Manual transmissionBrand new... More
- Fuel injected 190 HP Volkswagen VR6 engine- Toyota 5-speed transmission- British Racing Green exterior- New SS exhaust- 3 core aluminum radiator with electric... More
1967 Sunbeam Tiger Mk IA in British Racing Green with Black Vinyl Interior. This is an original car that has been very well preserved... More


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Sharpest mono boulder All bouldering8 routes in boulder
- 100% Boulder
- Grades: AU
- Ascents: 21
Did you know?
Did you know that you can create an account to record, track and share your climbing ascents? Thousands of climbers are already doing this.
Stats & downloads
Get directions to here using Google Maps
About 20m downhill from the top track is a boulder covered in mono's
Access issues inherited from Mt Yarrowyck
95% of the climbing at Yarrowyck is located on private property, but the land owner is a climber and has granted access to all climbers anytime. Please do not leave garbage around, leave gates as you find them and do not harass stock. Climbing at Yarrowyck is a privilege, not a right.
The other 5% is located on National Park land along the trail to the Aboriginal Rock Art site.
Left of pocket fest
Follow the wicked pockets, surprisingly good
Chin up on the twin mono's. A taste of 'Action Directe' in New England!
Up the arete on the west side
FA: Sam Mergatroyd?


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A protester holds a placard during a rally in support of Edward Snowden, the former National Security Agency contractor, in Hong Kong, China (left). Plus, a still from the BBC Horizon documentary, The Secret Life of Cats. Luke Casey / Bloomberg and bbc.co.uk
The UpTake: Where in the world is Edward Snowden? The flight of the nation's most prominent leaker of state secrets has us thinking about all things hidden and mysterious. All of today's links have a secretive air about them.
Links that got us thinking on Monday, June 24:
Scroll down to watch the BBC Horizon documentary: The Secret Life of the Cat.
Alex is a Brooklyn-based freelance journalist. He writes about media entrepreneurs and creatives for Upstart Business Journal.
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1. In a cast iron skillet, heat up some oil up to a couple of inches deep.
2. In the meanwhile, dredge the chicken strips, by coating it with first with flour, then egg and then the panko breadcrumbs.
3. Slide in the pieces of chicken into the hot oil and fry them until golden brown or until internal temperature reaches 180 degree F . Set them aside.
4. In a large mixing bowl, prepare the sauce by adding sesame oil, gochujang, soy sauce, honey and some hot water. Add few drops of the hot water at a time till the desired consistency is achieved.
5. Once the sauce is ready, add the fried chicken strips to it and toss until they are well coated with the sauce.
6. Serve the chicken strips garnished with a sprinkle of sesame seeds.


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Skip navigation.
Instructor of English
Vinson Hall 214
Tiffany Scarola is from Bolingbrook, Illinois. She holds a BA from Northern Illinois University, and a MA from DePaul University. Before Ms. Scarola obtained her MA she spent a year in Nanjing, China, where she was responsible for teaching English to 6th grade students at the Nanjing Foreign Language School. Upon returning to the states, Ms. Scarola attended DePaul University in Chicago, Illinois. Prior to her position at Northwestern, Ms. Scarola began work as an adjunct instructor at Waubonsee Community College in Aurora, Illinois. She was a part of the Department of College Readiness, where she taught writing and grammar classes for ESL/International students. While teaching at WCC, Ms. Scarola obtained a second position as an adjunct instructor at the College of DuPage in Glen Ellyn, Illinois. At COD, Ms. Scarola was responsible for teaching Composition I and Developmental Writing. Currently, Ms. Scarola is an Instructor of English and the Composition Coordinator for the English department. She also serves on the International Student Task Force, she is the faculty co-sponsor for Sigma Tau Delta, and is responsible for creating and teaching a composition class explicitly for the international student population.
English 0125: Writing & Literacy
English 1113: Composition I
English 1113: Composition I Special Topics: English for Academic Settings
English 1113L: Composition I Lab
English 1213: Composition II
English 3513: Technical Writing
MISSION STATEMENT: Northwestern Oklahoma State University provides quality educational and cultural opportunities to learners with diverse needs by cultivating ethical leadership and service, critical thinking and fiscal responsibility.


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Best Barley Soup
27 Jan 2010
|Whole barley
|1⁄4 Cup (4 tbs), washed
|Vegetable broth
|6 Cup (96 tbs), boiling (Homemade)
|Sliced carrots
|1 Cup (16 tbs)
|Diced celery
|1⁄2 Cup (8 tbs)
|Chopped onions
|1⁄4 Cup (4 tbs)
|Chopped peeled tomatoes
|2 Cup (32 tbs)
|Fresh peas
|1 Cup (16 tbs)
|To Taste
|Chopped parsley
|1⁄2 Cup (8 tbs)
1. Put the barley and broth in a heavy kettle, cover, and simmer until the barley is tender, about 1 hour. Add the remaining ingredients, with the exception of the parsley, and cook, covered, until the vegetables are barely tender.
2. Add the parsley, but do not cook any further.
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Best Barley Soup Recipe


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Old Town Home Prague
The building complex sprawls between two rustic, narrowly cobbled lanes, Tynska and Tynska ulicka, and in the heart of the building you will find a secluded courtyard. The complex boasts several venues of entertainment, including a pizzeria, hair salon, Bric-a-Brac shop, and our own Prague Trips & Tickets office. Even though all of our units are in the same building, the complex has multiple entrances, so some of our units are accessed from Tynska street and others from Tynska ulicka. Our Prague Trips & Tickets receptionist will guide you to your respective dorm or apartment.
Despite its age and the gloomy ending for some of it's previous owners, the building is clean and modern, keeping all of its guests comfortable. Our pleasantly sufficient rooms are gentle on the wallet yet in the best location. We offer prime choices of either private apartments or dorm rooms with ensuite bathrooms to provide for the needs of private groups or single travellers. The best choice for budget travelers wishing to stay in the most convenient spots!
Studio Apartment – Tynska ulicka 8
There is sleeping space for 4 people in the Studio. We provide a double bed accessed from padded steps in the sleeping loft. Below the loft, on the entrance level, there are 2 single beds and a kitchenette fully supplied with the utensils for cooking a quick, relaxing meal – electric kettle, microwave, refrigerator, silverware, cups, and dishes. The cozy table seats two people. Just down the hall from the Studio Apartment, is a newly refurbished private bathroom.
You enter the Studio Apartment from Tynska ulicka where there are restaurants, a tattoo parlor, and a sports bar.
1-Bedroom Apartment – Tynska 7
This Apartment Suite contains sleeping capacity for 4 occupants. There are 2 single beds in the first room sharing the space with a kitchenette. The kitchen area is complete with stove top, microwave, electric kettle, small refrigerator, and a dining table. All of the small wares necessary for cooking and eating are provided. The bedroom, has 2 single beds with accompanying lamp-stands, clothes rack, wardrobe, love seat, and shelves which allow you to unpack and live like at home instead of from a suitcase. There is a little washroom with toilet and tub off of the kitchenette.
The entrance is on Tynska, the main double wooden door. The apartment sits right above a little Gothic chapel in the building's courtyard which is recognizable by its stone arched doorway. You will go upstairs through a wrought iron gate to the first floor. Take the hall on the left, first door on the left to enter the apartment. The apartment's windows overlook the building's courtyard.
2-Bedroom Apartment – Tynska ulicka 8
This is a 2-bedroom apartment with both bedrooms accessible from the entry room. One bedroom holds 2 occupants with bed-side tables accompanying each bed. There is a large wardrobe to accommodate hanging clothes. The second bedroom is for 4 guests, 2 on the double bed and 2 on the single beds with its companion night stand. The entry room also contains access to the ensuite bathroom. There is a large fully equipped kitchen with a refridgerator and a dining table. A comfy couch and television make for a mini-living room adjoining the kitchenette. We supply free tea and coffee with individual creams. We provide fresh sheets and towels for the duration of your visit.
The entrance to the 2-Bedroom apartment is accessed from Tynska ulicka where you can find a tattoo parlor, restaurants, and a sport bar.
Large 2-Bedroom Apartment – Tynska ulicka 8
Our Large 2-bedroom apartment, located on the second floor without an elevator, is designed to sleep up to 6 guests comfortably. One bedroom has a double bed for 2 occupants and the second bedroom has 4 single beds. Both rooms provide bedside tables, wardrobes, or chest of drawers for unpacking. There is a fully equipped kitchen with eating area, wifi, computer, and fresh linens provided for all of our guests. This 2-bedroom apartment is accessed from Tynska ulicka and is on the second floor, without an elevator, so we don't recommend it for people with difficulties walking up stairs.
The entrance to the 2-Bedroom apartment is accessed from Tynska ulicka where you can find a tattoo parlor, restaurants, and a sport bar.
Availability & RatesTo view availability and prices please select dates and click 'Update Dates'
Apartment Facilities & Policies
- Airport transfers
- Breakfast not included
- Cafeteria
- Cash Only
- Child Friendly
- Cooker
- Cots available
- Credit Cards Accepted
- Free City Maps
- Free Internet Access
- Free Wifi
- Fridge/Freezer
- Internet Access
- Iron and Ironing Board
- Kitchen
- Linen Included
- Microwave
- Mini Supermarket
- Non Smoking
- Taxes included
- Tea/Coffee Making Facilities
- Towels Included
- Utensils
Cancellation PolicyThis property has a 3 day cancellation policy. Failure to cancel within this time will result in a cancellation charge equal to the first night of your stay.
Must-Read Apartment InfoThis property has specific Terms & Conditions.
Please Note! The Basic Studio apartment has a private external bathroom, just beside the apartment and not inside.
Studio Apartment is not suitable for families with children
The reception of the Old Town Home Prague is located in the tourist information center Prague Trips & Tickets at Tynska 7, which is at the same building on the ground level as the accommodation. You will receive and return keys in this office.
Our friendly staff is available to check you in and provide you with a variety of helps 10:00 to 21:00. If you will not be arriving within our opening hours, kindly notify us via email (firstname.lastname@example.com) or phone (+420 222 315 258) in advance to tell us what time you arrive. Due to Czech Law, each guest will have to fill out a registration card. Guest will pay for the accommodation costs immediately upon arrival.
Children, extra beds and pets:
Free! One child under 3 years stays free of charge when using existing beds.
There is a cot (baby travel bed) available upon request.
There is a child's booster seat for eating at the table available upon request.
Pets are not allowed.
DirectionsShow MapHide Map
How to find us by public transportation:
From the airport:
Buy a ticket in advance from a news stand, vending machine, or the actual bus driver and validate it in the orange device attached to a bus handle bar. The 34kc ticket is valid for 75 minutes and will give you enough time to reach us. Please note that if you buy the ticket on the bus from the driver it will cost 40kc and exact change is preferred.
You will take bus 119 from the airport to the last stop which is called Dejvicka (the same name as the metro station located there). Take the metro to the 3rd stop, Staromestska. After exiting the metro station onto Kaprova street, we are just a 5 minutes walk towards Old Town Square. You will find our office Prague Trips & Tickets at Tynska 7, Prague 1 behind the Tyn Cathedral. There you will check in at Old Town Home Prague.
From the main bus station:
Take the metro located next to the main bus station only one stop, from Florenc to Namesti Republiky. Or you can take bus 207 two stops from Florenc to Namesti Republiky.
From Namesti Republiky we are 5 minutes on foot to Prague Trips & Tickets, Tynska 7, Prague 1 where you will check in at Old Town Home Prague.
From main train station:
Take the metro one station, from Hlavni nadrazi to Muzeum where you will change lines from the C line to the A line. Then take the A line to Staromestska station (2 stops). You will walk 5 minutes toward Old Town Square to Prague Trips & Tickets, Tynska 7, Prague 1, found behind the Tyn Cathedral, to check in at Old Town Home Prague.
Located Near
Our accommodations are within a very short walking distance of the Old Town Square where you can see the Memorial Statue of John Huss and the Astronomical Clock dating from the 15th century located in the tower of Old Town Hall. There are also the noteable Kinsky Palace, now home of the National Gallery, the St. Nicolas Cathedral, and Tyn Cathedral to peruse while taking in the sights.
We are also very near several locally owned ammenities that make your stay feel more like home.


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I get asked every day, "Doctor, what should I eat? What should I be doing differently to try and stay out of this trouble?" This is very popular cocktail conversation. Is there a correlation between what we eat and colorectal cancer?
Well, we start with how colon cancer forms. We know that for almost all cancers, colon included, these are genetic mistakes that happen within the cell. We are all mutating all the time. Sometimes those mutations, we keep them around for the spice of life.
Sometimes those mutations though are so critical within our cell that the cell will shut down and stop and try to go over and fix that mutation before moving on. Sometimes, and probably by chance, that mutation happens in say the spell checker of the gene.
Now we don't get that little squiggly line under our DNA that notifies our cell that something is up, so it doesn't make a decision. It doesn't go over and fix it or let it be, if you will. We think most of cancer occurs because of those events.
Now, for the most part they are random. I have plenty of cardboard eating, marathon running patients. On the other hand, those folks who gamble a little bit more often have a higher chance that one of those unusual genetic mutations will happen.
We do believe diet plays a part in that. We're not really sure what it is about a high fiber low fat diet that's protective. It could be the high fiber. High fiber sort of waters down so to speak all that bad stuff we ate.
It also sort of has a transient…your stool moves through your bowel faster so it has less time to interact with the wall of the bowel. The low fat piece may have something to do with actually bile salts, believe it or not.
If you change your fat content in your diet, your liver changes the kind of bile salts it makes. They may be in fact different or more protective or less protective.
A high fiber low fat diet is our advice. A good balanced diet is the best thing you can do right now to stay out of my office.
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Results 1 to 3 of 3
Thread: Bronze Man
November 19th, 2007 #1
Bronze Man
Here's a fella donned in bronze I'd like to show ya all. *cough* This is my second proper attempt at working in photoshop, and hopefully it's not too bad.
Comments? Thanks.
- Similar Threads
- Art: Bronze
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- bronze woman
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- Art: Bronze Man
Hide this ad by registering as a memberNovember 19th, 2007 #2
I gotta say it bro but he kinda looks like C3PO. sorry if thats not what you wanted to hear. is this finished or are you going to render more?
November 20th, 2007 #3
In retrospect, I can see what you mean; I didn't even think once about the similarities while doing it. I do plan to work on a it a bit more, but I wanted to see someone else's opinion beforehand, and maybe get some pointers on how to improve it/techniques. At least I now have step 1; no more C-3PO sneaking in to the design. .


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The present invention provides a method for determining the amount of a target acid segment in a sample by polymerase chain reaction. The method involves the simultaneous amplification of the target nucleic acid segment and an internal standard nucleic acid segment. The amount of amplified DNA from each segment is determined and compared to standard curves to determine the amount of the target nucleic acid segment present in the sample prior to amplification. The method is especially preferred for determining the quantity of a specific mRNA species in a biological sample. Additionally, an internal standard is provided useful for quantitation of multiple mRNA species.
1. A method for quantifying a target nucleic acid segment in a sample, which method comprises the steps of:
(a) adding to said sample a predetermined initial amount of standard nucleic acid segment wherein said standard nucleic acid segment binds to same primers as are bound by said target nucleic acid segment in a reaction mixture;
(b) treating said sample under conditions suitable for carrying out a polymerase chain reaction, wherein said nucleic acids are rendered single-stranded and exposed to an agent for polymerization, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers, wherein said pair of primers is specific for both the target and standard nucleic acid segments, such that an extension product of each primer of said pair can be synthesized using separate strands of the target and standard segments as a template for synthesis, such that the extension product of one primer, when it is separated from the template strand, can serve as a template for the synthesis of the extension product of the other primer of said pair wherein said amplified target and standard segments are distinguishable by size or by the use of internal probes, wherein said internal probes may be differentially labeled for each of said amplified target and standard segments;
(c) separating the primer extension product from the templates on which they were synthesized to form single-stranded molecules;
(d) repeating steps (b) and (c) on the single stranded molecules produced in step (c) at least once, whereby each repeat of steps (b) and (c) is one amplification cycle;
(e) measuring the amounts of the amplified target and standard segments produced in step (d); and
(f) calculating from the amplified target and standard segments produced in step (d) the amount of said target nucleic acid segment present in the sample before amplification.
2. The method of claim 1 further comprising the step of preparing at least one dilution from the mixture of step (a) prior to step (b), whereby the amount of standard and target nucleic acid segments prior to said amplification reaction are varied.
3. The method of claim 1 wherein from 10 to 50 amplification cycles are performed, and wherein samples of said reaction mixture are removed and used to determine through which cycles the reaction is in exponential phase.
4. The method of claim 1 wherein said target nucleic acid segment is an mRNA molecule; said standard nucleic acid is a cRNA molecule; and said RNAs are reverse transcribed into cDNA molecules following step (a) and prior to step (b).
5. The method of claim 4 wherein the nucleic acid sequence of said cRNA molecule is synthesized using a plasmid template selected from the group AW108 and AW109.
6. The method of claim 4 wherein the pair of oligonucleotide primers of step (b) is labeled, and the amounts of amplified target and standard segments produced are measured according to step (e) by determining the amount of label incorporated into each of said amplified nucleic acid segments.
7. The method of claim 6 wherein said target nucleic acid segment is contained within an mRNA sequence, wherein said mRNA sequence encodes a lymphokine.
8. The method of claim 6 wherein said target nucleic acid segment is contained within a nucleic acid sequence, wherein said nucleic acid sequence encodes a protein selected from the group consisting of TNF, M-CSF, PDGF-A, PDGF-B, PDGF-R, apo-E, LDL-R, HMG, IL-1, IL-2, LPL, G-CSF, GM-CSF, aFGF, bFGF, c-fms, TGF-β, LFA-1, IL-2Rα, α-actin, desmin, β-actin, IL-6, IFN-α, IFN-γ, IL-6R, PDGF-αR, IL-2Rβ, IL-3, IL-4, and HIV proteins.
9. The method of claim 8 wherein said sample is a human blood cell sample or a human arterial vessel sample.
10. The method of claim 8 wherein said pair of oligonucleotide primers are selected from the group consisting of:
______________________________________5'-CAGAGGGAAGAGTTCCCCAG-3',5'-CCTTGGTCTGGTAGGAGACG-3';5'-GAACAGTTGAAAGATCCAGTG-3',5'-TCGGACGCAGGCCTTGTCATG-3';5'-CCTGCCCATTCGGAGGAAGAG-3',5'-TTGGCCACCTTGACGCTGCG-3';5'-GAAGGAGCCTGGGTTCCCTG-3',5'-TTTCTCACCTGGACAGGTCG-3';5'-TTCCTGGCAGGATGCCAGGC-3',5'-GGTCAGTTGTTCCTCCAGTTC-3';5'-CAATGTCTCACCAAGCTCTG-3',5'-TCTGTCTCGAGGGGTAGCTG-3';5'-TACCATGTCAGGGGTACGTC-3',5'-CAAGCCTAGAGACATAATCATC-3';5'-GTCTCTGAATCAGAAATCCTTCTATC-3',5'-CATGTCAAATTTCACTGCTTCATCC-3';5'-AAACAGATGAAGTGCTCCTTCCAGG-3',5'-TGGAGAACACCACTTGTTGCTCCA-3';5'-GAATGGAATTAATAATTACAAGAATCCC-3',5'-TGTTTCAGATCCCTTTAGTTCCAG-3';5'-TGACCACCCAGCCATCCTTC-3',5'-GAGGAGGTGTTGACTTCATTC-3'; and5'-GAGATTTCTCTGTATGGCACC-3',5'-CTGCAAATGAGACACTTTCTC-3'.______________________________________
This application is a continuation-in-part of copending U.S. Ser. No. 396,986, filed Aug. 21, 1989 now abandoned.
1. Field of the Invention
The present invention relates to the quantitative determination of a particular nucleic acid segment in a sample. The invention is particularly useful for determining the quantity of specific mRNA molecules in a biological sample. The method is therefore especially applicable in the field of medical diagnostics but can also be applied in the fields of genetics, molecular biology, and biochemistry.
2. Description of Related Disclosures
U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose methods for carrying out the polymerase chain reaction (PCR), a nucleic acid amplification method, and for using PCR in the detection of specific nucleotide sequences. European Patent Office Publication (EPO) No. 258,017 describes Taq polymerase, a preferred DNA polymerase for use in PCR. These publications are incorporated by reference herein.
PCR methods have widespread applications in genetic disease diagnosis (see Wu et al., 1989, Proc. Natl. Acad. Sci. USA 86:2757-2760 and Myerswitz, 1988, Proc. Natl. Acad. Sci. USA 85:3955-3959), as well as disease susceptibility and cancer diagnosis (see Horn et al., 1988, Proc. Natl. Acad. Sci. USA 85:6012-6016; Todd et al., 1987, Nature 329:599-604; Kawasaki, 1988, Proc. Natl. Acad. Sci. USA 85:5698-5702; and Neri et al., 1988, Proc. Natl. Acad. Sci. USA 85:9268-9272). However, these uses have provided only qualitative results by, for example, detecting unique mRNA transcripts from abnormal cells in a background of normal cells.
An attempt to use PCR for quantitative studies of mRNA level for thymidylate synthase in tumors has been published (see Kashani-Sabet, 1988, Cancer Res. 48:5775-5778). However, this study provides only relative comparisons of amounts of mRNA in biological samples. It has been much more difficult to quantitate the absolute amount of specific mRNA without an internal standard of known concentration. Other methods have been described for quantitating nucleic acid species by using PCR to co-amplify a second, unrelated, template cDNA (see Chelly et al., 1988, Nature 333:858-860 and Rappolee et al., 1988, Science 241:708-712). The use of an unrelated cDNA standard also necessitates the use of a second set of oligonucleotide primers, unrelated to the specific target mRNA.
Because amplification is an exponential process, small differences in any of the variables which control the reaction rate, including the length and nucleotide sequence of the primer pairs, can lead to dramatic differences in the yield of PCR product. Analyses which use two sets of unrelated primers, therefore, can only provide a relative comparison of two independent amplification reactions rather than an absolute measure of mRNA concentration.
Gilliland et al. (J. Cellular Biochemistry, UCLA Symposia on Molecular and Cellular Biology, Apr. 3-21, 1989, Abstract WH001) describe alternative approaches to mRNA quantitation to avoid some of the problems associated with unrelated templates as amplification standards. However, the Gilliland et al. suggestions have other inherent limitations. One approach requires mapping of genomic introns and exons for the gene corresponding to a specific target mRNA. Gilliland et al. also proposes an alternative approach using site directed mutagenesis to construct an internal standard, which causes the formation of heteroduplexes following amplification. These heteroduplexes result in an over estimation of the amount of target sequence present in the original sample. Smith et al. (Smith et al., 1989, J. Immunol. Meth. 118:265-272) have used an RNA dot blot assay to asses quantitatively the expression level of the two IL-1 mRNAs in human macrophages. Smith et al. reported that the level of sensitivity for IL-1α mRNA was approximately 107 molecules by his method, and IL-1α mRNA was undetected in uninduced macrophages. The present invention provides a quantitation method which can readily measure 104 molecules and readily detects IL-1α mRNA in uninduced as well as induced macrophages in a sample assay. This 1000 fold increase in sensitivity represents an important advance in quantitative analysis for clinical and research purposes.
There remains a need for a method to quantitate directly, accurately, and reproducibly the amount of a specific nucleic acid segment in a sample. The availability of quantitative PCR will provide valuable information in a number of research areas. More particularly, the invention provides critical information in disease diagnosis and cancer therapy. For example, a reliable, sensitive, quantitative analysis can be critical in determining the extent of induction of mRNA synthesis in response to exogenous stimuli. The present invention overcomes the numerous limitations inherent in the attempts of others in this field, and thus provides means for accurately quantifying the amount of a nucleic acid segment in a biological sample.
The present invention provides a method for quantifying a target nucleic acid segment in a sample, which method comprises the steps of:
(a) adding to said sample an amount of standard nucleic acid segment;
(b) treating said sample under conditions suitable for carrying out a polymerase chain reaction, wherein said nucleic acids are rendered single-stranded and exposed to an agent for polymerization, deoxynucleoside 5' triphosphates, and a pair of oligonucleotide primers, wherein said pair of primers is specific for both the target and standard nucleic acid segments, such that an extension product of each primer of said pair can be synthesized using separate strands of the target and standard segments as a template for synthesis, such that the extension product of one primer, when it is separated from the remplate strand, can serve as a template for the synthesis of the extension product of the other primer of said pair;
(c) separating the primer extension product from the templates on which they were synthesized to form single-stranded molecules;
(d) repeating steps (b) and (c) on the single stranded molecules produced in step (c) at least once, whereby each repeat of steps (b) and (c) is one amplification cycle;
(e) measuring the amounts of the amplified target and standard segments produced in step (d); and
(f) calculating from the amplified target and standard segments produced in step (d) the amount of said target nucleic acid segment present in the sample before amplification.
The present invention also provides a plasmid useful for providing an internal standard for quantitation of target nucleic acid segments, said plasmid comprising a DNA sequence, said DNA sequence further comprising sequences which are identical to DNA sequences contained within said target nucleic acid segments.
The present invention also provides kits for the quantitation of specific nucleic acid segments in a biological sample.
FIG. 1 shows the positions of the 5' primers and 3' primers of Table I as they are arranged in plasmids AW108 and AW109. Other features are shown as they relate to the present invention.
In FIG. 2A-C, the amount of IL-1α mRNA present in lipopolysaccharide (LPS) induced and uninduced macrophages was determined using the IL-1α primer pair.
FIG. 2A depicts an ethidium bromide stained acrylamide gel wherein the amplified standard and target DNA segments are visible.
FIG. 2B plots the amounts of standard and target IL-1α PCR product DNA produced against template concentrations.
FIG. 2C shows a plot of the amounts of standard and template IL-1α PCR product DNA produced versus the number of amplification cycles.
FIG. 3 shows the results of a Northern blot containing samples of AW108 cRNA, and RNA isolated from LPS induced macrophages. The blot was probed with the IL-1α 3' primer.
FIG. 4 shows the efficiency of amplification for different primer sets using the same cRNA template under the same conditions.
The present invention provides a method for determining the absolute amount of a nucleic acid segment in a sample. The method involves amplification, by a polymerase chain reaction, of two different segments of nucleic acid combined in one reaction mix. The two segments include a target segment and an internal standard segment. The internal standard is amplified using the same oligonucleotide primer pair as the target nucleic acid; however, the two nucleic acid segments yield amplified products which are distinguishable by size.
The standard segment is present in a known amount. Following amplification, the amount of each of the two polymerase chain reaction products is measured, and the amount of the target segment present in the original sample is quantitated by extrapolating against a standard curve. In addition, the internal standard described herein contains primer sequences for multiple genes, so that the same standard can be used to quantitate a number of different nucleic acid segments of interest.
The present invention has particular utility in providing a rapid, sensitive, and reliable method for accurately determining the quantity of low abundance, specific mRNAs present in a sample containing less than 0.1 ng of total RNA. The method provides an approach powerful enough to enable a measurement of heterogeneity of expression levels of specific mRNAs within particular subpopulations even at the single cell level.
By co-amplification of the target nucleic acid and the internal standard nucleic acid, variable effects are internally controlled and affect the yield of PCR product equally for target and standard nucleic acids. Numerous variables influence the rate of the PCR reaction. Such variables may include the concentrations of polymerase, dNTPs, MgCl2, nucleic acid templates, and primers, as well as the rate of "primer-dimer" formation and tube-to-tube variations.
The amount of the target nucleic acid segment present in the sample prior to amplification is determined using a standard curve. The standard curve is generated by plotting the amount of the standard segment produced in a polymerase chain reaction against varying, but known, amounts of the RNA present before amplification. For accuracy, the amount of standard segment present before amplification is varied by serial dilution of the co-amplification reaction mix. The amount of target segment produced in the polymerase chain reaction is then compared to the standard curve to determine the amount of target segment present in the sample prior to amplification. Alternatively, the standard curve may be generated by plotting the amount of standard and target segments produced against the number of amplfication cycles. To ensure accuracy, it is preferred that the number of amplification cycles is varied by removing aliquots from one co-amplification reaction mixture after different numbers of amplification cycles have been completed.
The method of the invention is far superior to determinations of the amount of a nucleic acid segment in a sample as a relative, rather than absolute, amount. Further, the method is far more accurate than when an absolute amount is derived by employing a second set of oligonucleotide primers to amplify the standard, wherein that set of primers is different from the set used to amplify the target segment.
The method of the present invention is useful for quantifying a target RNA or DNA molecule. For determining an amount of DNA present in a sample, amplification methods described herein can be applied directly. As the examples disclosed below will demonstrate, the present invention is also useful in determining the amount of a specific mRNA in a sample of total RNA. The internal standard nucleic acid segment is provided on a DNA plasmid. The presence of an appropriately placed T7 polymerase promoter or another suitable promoter, such as the SP6 promoter, allows the plasmid to be used as a template for cRNA synthesis. As defined herein for the purpose of the present invention, the term "cRNA" refers to a ribonucleic acid segment synthesized from a DNA template by an RNA polymerase. Further, the plasmid may contain a polyadenylation sequence at the 3' end to facilitate purification and subsequently quantitation of the in vitro synthesized cRNA. As described in the preferred embodiments, the DNA template is either plasmid AW108 or AW109, and the RNA polymerase is T7 polymerase. In one embodiment AW108 cRNA is synthesized as a sense strand from pAW108 by T7 polymerase. The structure of pAW108 is shown in FIG. 1. The primer array as shown in FIG. 1 is identical for both pAW108 and pAW109. The cRNA molecule then serves as the internal standard template for reverse transcription by the DNA polymerase, reverse transcriptase. Reverse transcriptase generates a cDNA transcript from an RNA template. The preferred embodiment of the invention, the internal standard cRNA, is synthesized as a sense strand. Following reverse transcription of the target mRNA and the standard cRNA, PCR is then performed.
As will be obvious to those skilled in the art, numerous methods are known for constructing plasmids useful in the method of the present invention. Higuchi, 1988, Nucleic Acids Research 16:7351-7367 and Ho, 1989, Gene 77:51-59 describe two methods for engineering novel plasmids which incorporate desired synthetic DNA sequences. Alternatively, synthetic DNA segments can be inserted via restriction enzyme digestion and ligation with an appropriately treated parent plasmid or phage vector. The internal standard of the preferred embodiment, pAW108, contains multiple primer sets which allow a single cRNA standard to be used to quantitate a number of different mRNAs. The presence of unique restriction enzyme sites in the pAW108 plasmid provides the flexibility to add new primer sets to the plasmid. The unique BamHI site is used to linearize the plasmid to produce a linear template for reverse transcription. A deposit of E. coli containing plasmid AW108 has been deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md. according to the terms of the Budapest Treaty. A deposit of E. coli containing plasmid AW109 has also been deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md. according to the terms of the Budapest Treaty.
Plasmid AW108 is derived from pcDV1 and pL1 which are disclosed in Okayama and Berg, 1983, Mol. Cell Biol. 3:280-289. The SV40 promotor region from pLI was inserted into pcDV1 as directed in the referenced article. The T7 promoter, synthetic oligonucleotide sequences, and a polyadenylation region from the IL-1α gene were then inserted to provide the AW108 plasmid as an internal standard for the quantitation of twelve specific mRNAs. The plasmid was transformed into E. coli and grown in Luria Broth with ampicillin at 50 μl/ml added.
Plasmid AW108 was subsequently used as the starting material to construct pAW109. A culture of E. coli containing pAW108 was grown, and plasmid DNA was purified by standard means. The plasmid was digested with BamHI and BglII restriction endonucleases, and the 1 kb fragment was purified. This fragment contained the 5' and 3' primer arrays shown in FIG. 1 as well as the polyadenylation sequence. Plasmid pSP72 (Promega Biotec, Madison, Wis. contains a T7 promoter adjacent to a polylinker to facilitate cloning. The plasmid also contains the ampicillin resistance gene.
The BglII-BamHI fragment from pAW108 was ligated into BglII and BamHI cleaved pSP72. Both of these are unique restriction sites within the polylinker region. The ligation mixture was used to transform E. coli DH5α, and resultant ampicillin resistant colonies were selected. The plasmid was assayed for the correct orientation of the BglII-BamHI insert. The resulting plasmid, pAW109, is suitable as an internal standard for mRNA quantitation.
As will be obvious to those skilled in the art, numerous other plasmids are available for insertion of desired DNA sequence to provide an internal standard useful in the present invention. Generally, the methods for transformation of such plasmids into a suitable host strain, propagation of the transformed host, and preparation of plasmid DNA as required for practice of the invention can be found in Maniatis et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1985.
As used herein, the term "5' primer" refers to an oligonucleotide comprising a sequence identical to the sequence contained within the sense strand of a target nucleotide segment. As used herein the term "3' primer" refers to an oligonucleotide comprising a sequence complementary to a sequence contained within the sense strand of the same target nucleotide segment. Thus, a 3' primer useful in the method of the present invention will hybridize to an mRNA, cRNA, or DNA template. It is further descriptive of the 3' and 5' primers that for both the internal standard cRNA and the target mRNA segment, the region of 3' primer hybridization is located 3' to the region of 5' primer hybridization.
The 3' and 5' primers function in the method of the present invention as follows: the 3' primer primes DNA synthesis in a PCR reaction to produce an antisense DNA strand, which provides a template for second strand DNA synthesis when the 5' primer is included in the PCR reaction. Such a 5' and 3' primer are referred to herein as a "primer pair."
In the preferred embodiment, most members of a primer pair are designed to span two exon-intron junctions within the gene encoding each target mRNA. In this way the primers will only hybridize effectively to the desired target mRNA. Thus, small amounts of contaminating genomic DNA in a biological sample will not effect accurate quantitation of the target mRNA.
Thus, a primer pair will function in a PCR reaction to amplify a segment of nucleic acid having a primer sequence identical to a DNA segment contained within the standard nucleic acid, i.e., as illustrated here, plasmids AW108 and AW109. As described herein, both plasmids contain a DNA sequence which comprises the DNA sequence of twelve primer pairs arranged as follows: DNA identical in sequence to the 5' primers of twelve target mRNAs is followed by the complementary DNA sequence of the 3' primers for the same twelve target mRNAs (FIG. 1). The primer pair DNA sequence within pAW108 and pAW109 corresponds to mRNAs encoding tumor necrosis factor (TNF), macrophage-colony stimulating factor (M-CSF), platelet-derived growth factor A (PDGF-A), platelet-derived growth factor B (PDGF-B), low density lipoprotein receptor (LDL-R), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), type beta platelet-derived growth factor receptor (PDGF-βR), and lipoprotein lipase (LPL). The primer pairs useful for amplifying the internal standard provided by AW108 or AW109 cRNA in the practice of the method of the invention are depicted in Table I.
TABLE I__________________________________________________________________________Oligonucleotides of 12 Target Genes' 5' Primers and 3' Primers Size of PCRmRNA Product (bp)Species5' Primers 3' Primers mRNA cRNA__________________________________________________________________________TNF 5'-CAGAGGGAAGACTTCCCCAG-3' 5'-CCTTGGTCTGGTAGGAGACG-3' 325 301M-CSF5'-GAACAGTTGAAAGATCCAGTG-3' 5'-TCGGACGCAGGCCTTGTCATG-3' 171 302PDGF-A5'-CCTGCCCATTCGGAGGAAGAG-3' 5'-TTGGCCACCTTGACGCTGCG-3' 225 301PDGF-B5'-GAAGGAGCCTGGGTTCCCTG-3' 5'-TTTCTCACCTGGACAGGTGG-3' 217 300apo-E5'-TTCCTGGCAGGATGCCAGGC-3' 5'-GGTCAGTTGTTCCTCCAGTTC-3' 270 301LDL-R5'-CAATGTCTCACCAAGCTCTG-3' 5'-TCTGTCTCGAGGGGTAGCTG-3' 258 301HMG 5'-TACCATGTCAGGGGTACGTC-3' 5'-CAAGCCTAGAGACATAATCATC-3' 246 303IL-1α5'-GTCTCTGAATCAGAAATCCTTCTATC-3' 5'-CATGTCAAATTTCACTGCTTCATCC-3' 420 308IL-1β5'-AAACAGATGAAGTGCTCCTTCCAGG-3' 5'-TGTTTCAGATCCCTTTAGTTCCAG-3' 388 306IL-2 5'-GAATGGAATTAATAATTACAAGAATCCC-3' 5'-TCCAGAACACCACTTGTTGCTCCA-3' 222 305PDGF-R5'-TGACCACCCAGCCATCCTTC-3' 5'-GAGGAGGTGTTGACTTCATTC-3' 228 300LPL 5'-GAGATTTCTCTGTATGGCACC-3' 5'-CTGCAAATGACACTTTCTC-3' 277 300__________________________________________________________________________ TNF, tumor necrosis factor; MCSF, macrophagecolony stimulating factor; PDGFA, plateletderived growth factor A; PDGFB, plateletderived growth factor B; apoE, apolipoprotein E; LDLR, low density lipoprotein receptor; HMG, 3hydroxy-3-methylglutaryl coenzyme A reductase; IL1α, interleukin1α; IL1β, interleukin1β; IL2, interleukin2; PDGFR, type β plateletderived growth factor receptor; LPL, lipoprotein lipase.
Other mRNA targets which may be readily quantitated in biological samples by the present invention include, but are not limited to, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), acidic-fibroblast growth factor (aFGF), basic-fibroblast growth factor (bFGF), c-McDonough feline sarcoma (c-fms), transforming growth factor-β (TGF-β), leukocyte adhesion protein-1 (LFA-1), interleukin-2 receptor-α (IL-2Rα), alpha-actin, desmin, β-actin, interleukin-6 (IL-6), interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-6 receptor (IL-6R), platelet derived growth factor-α receptor (PDGF-αR), interleukin-2 receptor-β (IL-2Rβ), interleukin-3 (IL-3), and interleukin-4 (IL-4) as well as human immunodeficiency virus (HIV). Examples of primer pairs useful for the detection and measurement of expression of these RNAs are exemplified by the oligonucleotide sequences shown in Table II.
TABLE II__________________________________________________________________________G-CSF 5' GGTGAGTGAGTGTGCCACCT3', 5' GTTCTTCCATCTGCTGCCAG3' ;GM-CSF 5' CACTGCTGCTGAGATGAATGAAACAG3', 5' GCACAGGAAGTTTCCGGGGTTGG3' ;aFGF 5' TCCTTCCGGATGGCACAGTC3', 5' CATTTGGTGTCTGTGAGCCG3' ;bFGF 5' GACCCTCACATCAAGCTACAAC3', 5' GGAAGAAAAGTATAGCTTTCTGC3' ;c-fms 5' CAAGTATAAGCAGAAGCCCAAGTAC3', 5' GAGGGTCTTACCAAACTGCAGG3' ;TGF-β 5' CATCAACGGGTTCACTACCG3', 5' TCCGTGGAGCTGAAGCAATAG3' ;LFA-1 5' GAGTGCCTGAAGTTCGAAAAGG3', 5' CACACACTCTCGGCTCTCATC3' ;IL-2Rα 5' GCTGCCAGGCAGAGCTCTGTGACG3', 5' GTTCCGAGTGGCAGAGCTTGTGC3' ;α-actin 5' GCACAACTGGCATCGTGCTG3', 5' AGACTCCATCCCGATGAAGG3' ;desmin 5' AGGAGAGCCGGATCAACCTTC3', 5' TCGCTGACGACCTCTCCATC3' ;β-actin 5' CCTTCCTGGGCATGGAGTCCTG3', 5' GGAGCAATGATCTTGATCTTC3' ;IL-6 5' CCTTCTCCACAAGCGCCTTC3', 5' GGCAAGTCTCCTCATTGAATC3' ;IFN-α 5' AGCTGCAAGTCAAGCTGCTC3', 5' TCCCAAGCAGCAGATGAGTC3' ;IFN-γ 5' GAAGAATTGGAAAGAGGAGAGTGACAGAAA3', 5' CATTCAAGTCAGTTACCGAATAATTAGTCAG3' ;IL-6R 5' CATTGCCATTGTTCTGAGGTTC3', 5' AGTAGTCTGTATTGCTGATGTC3' ;PDGF-αR 5' CTGGATGAGCAGAGACTGAG3', 5' AGGAAGCTGTCTTCCACCAG3' ;IL-2Rβ 5' TTTCAGGTGCGGGTCAAGCCTCTG3', 5' AGTAACCCTGGTTGGTGAAGCAGC3' ;IL-3 5' CATGAGCCGCCTGCCCGTCC3', 5' GGTTATTTTCCATCAGAATG3' ;IL-4 5' CTCACCTCCCAACTGCTTCCC3', 5' GTGGAACTGCTGTGCAGTCGC3' ; andHIV 5' AGTGGGGGGACATC3', 5' TTTGGTCCTTGTCTTATG3'.__________________________________________________________________________
The PCR product from each primer set within pAW108 and pAW109 is 300-308 base pairs (bp), depending on the particular primer pair used. The 300-308 bp segment length of the illustrated example does not impose a limitation to the design of any internal standard. It is only necessary that the standard segment length is designed to be different in size from the PCR products of the target mRNAs and that the segment lengths be within the detection limits inherent in the analytical system preferred (for example, acrylamide gel electrophoresis, agarose gel electrophoreses, or other chromatographic means). The size difference between the PCR amplification products permits easy separation of the internal standard cRNA amplification product from the target mRNA amplification product by, for example, gel electrophoresis. The unique BamHI site is used to linearize the AW108 or AW109 plasmid to produce cRNA transcripts. Such transcripts are useful for quantitation of a number of different specific mRNAs in, for example, treated and untreated samples. This method can be used to provide a transcriptional phenotype of a treated or untreated cell or tissue and thus provides for numerous clinical and research applications.
The cRNA and the target mRNA are reverse transcribed in the same reaction. In this way, the cRNA serves not only as a standard for mRNA quantitation, but also provides an internal mRNA control for the reverse transcription reaction. Reverse transcriptase requires a primer to initiate cDNA synthesis using an RNA template. In the practice of the present invention, this will be an oligonucleotide primer which hybridizes to both the standard cRNA and the target mRNA. The primer may be identical in sequence to the 3' primer used for PCR amplification of that target mRNA. Alternatively, the primer for the reverse transcription reaction may be an oligonucleotide which hybridizes to the mRNA and cRNA at a position distal to the sequence of the 3' amplification primer, for example, oligo (dT). Thus, the resultant cDNA contains within it a sequence identical to the sequence of the 3' amplification primer. In the preferred embodiment disclosed herein, AW108 cRNA, as well as AW109 cRNA, contain a polyadenylation sequence at the 3' end, and oligo (dT) is used as a primer for reverse transcription of the cRNA and mRNA templates. Additionally, oligo (dT) permits amplification of more than one target sequence from the same reverse transcriptase reaction mix.
The same primers are used in the PCR amplification of both the standard and target templates; therefore, there are no primer efficiency differences between amplification of the standard and the target RNAs. When dilution series of mixtures of the target mRNA and internal standard cRNA are amplified in the same tube, and the reaction is terminated in the exponential phase of the amplification, the amount of target mRNA that was present in the sample prior to amplification can be determined by extrapolating against the internal standard cRNA standard curve. The amount of DNA produced is plotted against the amount of starting material for both the standard and the target. The standard curve allows extrapolation of the target data to determine the amount of target in the starting material. This value may be expressed as molecules of target mRNA/ng total RNA. Alternatively, it may be determined as of percentage or an amount by weight, or as a copy number.
Alternatively, a method is provided for determining the amount of target mRNA by varying the number of amplification cycles. The amount of amplified products produced is plotted against the number of amplification cycles for both the standard and target segments. The plotted data illustrates that portion of the reactions wherein the rate of amplification is exponential. Therefore, a ratio of products formed can be equated to a ratio of starting materials to determine the initial amount of target segment present. This is done according to the formula: ##EQU1## where No is the initial amount of material, and N is the amount of amplified product produced.
In another embodiment of the present invention, a third primer array is inserted into the internal standard plasmid between the 5' primer array and the 3' primer array. The third oligonucleotide array is comprised of a series of synthetic sequences wherein there is one sequence corresponding to each RNA for which the plasmid contains a 5' and 3' primer pair. This array is designed such that for each target RNA to be quantitated, the amplified product will contain within it a sequence identical to a portion of the third oligonucleotide array. Thus, both the amplified target and amplified standard DNA segments contain an identical internal segment providing a probe hybridization site, whereby for each primer pair, an oligonucleotide probe is useful to detect the amplified target as well as the amplified standard DNA.
Where a third oligonucleotide array is included in the standard plasmid, the PCR reaction can be carried out without the use of label. It is preferred that the reverse transcription and amplification reactions are carried out in separate tubes for each of the standard and target templates, rather than as a co-amplification. Following amplification, amount of product is quantiated by use of a dot blot format employing a single-stranded oligonucleotide probe which has a sequence corresponding to the internal sequence provided by the third primer array.
As an illustrative example of the present invention, the AW108 internal standard was used to determine the amount of several lymphokine mRNAs, including IL-1α mRNA, isolated from lipoplysaccharide (LPS)-induced and control cultures of human macrophages. Lymphokine mRNA levels were also measured in human atherosclerotic plaque tissue.
As provided by the present invention, target mRNA is quantified most accurately by using an internal standard having, in part, the same sequence as the target itself. Quantification of mRNA sequences by PCR amplification using an unrelated template as an internal standard provides only comparative data because of differences in efficiency between the primer pairs for the standard and the target mRNAs. This is inherent in the amplification process because PCR amplification is an exponential process. The extent of amplification (N) is given by the equation: N=No (1+eff)n where No is the initial amount of material, eff is the efficiency, and n is the cycle number. Thus, small differences in efficiency lead to large differences in the yield of PCR product and result in a misrepresentation of the amount of template present in a biological sample. Further, differences in primer efficiency are difficult parameters to regulate for quantitative analyses. The present invention overcomes these problems.
The significant contribution of primer efficiency in the accurate quantitation of a nucleic acid segment is underscored in an example below. AW108 cRNA was used as the template for PCR amplification of several different primer sets. The efficiency of amplification by these different primer sets, under the same PCR conditions, varies over a range of several orders of magnitude. This invention addresses itself to this issue, which is clearly critical in any attempt to quantitate mRNA expression by PCR, and overcomes the problem of primer efficiency by using the same primers for amplification of the target mRNA and the internal standard cRNA.
The present invention requires that the amplification of the standard and target segments of nucleic acid be carried out in the same reaction. In the preferred embodiment of the present invention, the reverse transcriptase reaction of the standard cRNA and target mRNA is also carried out in the same reaction. Those skilled in the art will recognize from the foregoing that one could quantitate a target nucleic acid by performing the standard and target reverse transcriptase and amplification reactions separately. However, the accuracy of such a method is dependent on the degree to which the reverse transcription and amplification steps proceed with similar efficiency for both amplifications. By performing both reverse transcriptase reactions in the same tube and both amplification reactions in the same reaction tube, one ensures excellent accuracy.
The amount of an amplified DNA fragment in a given sample can influence amplification efficiency. When a high template concentration is used, or occurs as a result of the PCR amplification, phenomena can occur which are limiting factors for efficient amplification. Such phenomena include substrate saturation of enzyme, product inhibition of enzyme, incomplete product strand separation, and product strand reannealing. These problems are readily avoided, however, by an initial titration of the specific target mRNAs to find the range of concentrations that gives exponential amplification over a defined range of cycle numbers. Accordingly, to obtain reliable quantitative evaluation of specific mRNA using the described invention, the range of concentrations for both the standard and target templates, as well as the number of amplification cycles, should be such that the reactions remain within the exponential phase.
Thus, in the preferred embodiment, the reaction conditions described make use of 50 ng-1 μg of total cellular RNA combined with approximately 2�102 -2�107 molecules of cRNA. As little as 50 pg cellular RNA is also suitable for purposes of the present invention. In the example described, as few as 1�104 molecules of IL-1α are detected. It is not necessary that mRNA be purified from a total RNA preparation in order to employ the method of the invention.
Samples suitable for analysis by this method may be of human or non-human origin; they may be derived from cultured samples, or isolated from dissected tissue or from cells of immunologically defined phenotype. The latter can be obtained by monoclonal antibody staining and fluorescence-activated cell sorter (FACS) isolation of enzyme-dissociated cells or by removal of specific areas from immunohistochemically stained slides. This will permit definitive identification of the cell types producing specific mRNAs.
The amount of amplified DNA generated in the method of the present invention can be measured in different ways. For instance, labeled primers wherein one or both members of any primer pair is labeled, or labeled nucleotides, can be used in PCR, and the incorporation of the label can be measured to determine the amount of amplified DNA. The label can be isotopic or non-isotopic. Alternatively the amount of amplified product can be determined by electrophoresis and visualization of the amplified product by staining or by hybridization with a labeled probe. Densitometry can be used to calculate the amount of product on a stained gel, or by extrapolation from an autoradiograph when labeled probe is used. When a labeled probe is used, the probe should be present in excess of the amplified product. In one such embodiment of the invention, primers are isotopically labeled and the resultant amplified products are electrophoresed on an acrylamide gel. The region where the product is expected to migrate is excised, and the amount of label present is determined by Cerenkov counting. The amount of label present is plotted versus the amount of known starting material.
The method of the invention requires that the amplified amounts of a template and standard segment produced in a single polymerase chain reaction be determined. Thus, the method requires that the amplified template segment be distinguishable from the amplified standard segment. If the segments are of different sizes, then it is relatively simple to distinguish one amplified segment from the other, i.e., the amplified products can be readily separated by gel electrophoresis. The present invention does not require that the amplified product be of different sizes, however, for other methods can be utilized to distinguish one amplified segment from another. For instance, the internal probe specific for one segment can be labeled differently than the internal probe specific for the other segment.
The quantitative method described herein is useful for analyses of in vivo biological samples. As is illustrated in the following example, quantitative PCR analysis of PDGF-A and B chain mRNA a human atherosclerotic lesion versus a normal blood vessel emphasizes the sensitivity of this approach in investigating the biology of cells and tissues in vivo. For example, when the present method was used to measure IL-1α and IL-1β mRNAs in atherosclerotic tissue, the results suggested that there may be inflammatory or immunological components in the pathogenesis of the disease.
Due to its high sensitivity, speed, and accuracy, the present quantitative PCR method can be used to study gene expression in a more extensive way than has been possible to date, allowing quantitative measurements of gene expression in a very small number of cells and from small amounts of tissue samples available from in vivo sources, such as biopsies. This technique can also provide information on changes in expression level of specific RNA molecules which may be valuable in the diagnosis and analysis of, for example, infectious disease states, cancer, metabolic disorders, and autoimmune diseases.
It will be apparent to those skilled in the art that the method of the present invention is amenable to commercialization as a kit for the quantitation of one or more nucleic acids in a sample. For example, in its simplest embodiment, such a kit would provide an internal standard and an appropriate oligonucleotide primer pair. In another embodiment, a kit may contain an internal standard, an appropriate oligonucleotide primer pairs, a DNA polymerase, a RNA polymerase, a reverse transcriptase, nucleotide triphosphates, restriction enzymes, buffers for carrying out cRNA and cDNA synthesis, restriction enzyme digests, and amplification by PCR. Further, the kits may contain a thermostable DNA polymerase; for example, the thermostable DNA polymerase Taq isolated from Thermus aquaticus as an agent of polymerization.
The method of the invention is exemplified below, but those skilled in the art will recognize the present invention is broadly applicable and in no way limited to the specific embodiments described below.
A synthetic gene was constructed using a technique of oligonucleotide overlap extension and amplification by PCR. The procedure used was similar to that described by Ho et al. for use in site-directed mutagenesis (Ho et al., 1989, Gene 77: 51-59). After construction, the synthetic gene was subcloned into an Okayama-Berg vector containing the T7 polymerase promoter and a polyadenylated sequence. The resulting plasmid, AW108, is shown in FIG. 1. This plasmid was used as a template for transcription by T7 polymerase according to the transcription protocol of the manufacturer (Promega Biotec, Madison, Wis. The resulting AW108 cRNA product was purified by oligo(dT) chromatography and gel electrophoresis. Alternatively, pAW109 was used to prepare a cRNA standard. The cRNA product was purified by selective elution using the QIAGEN-tip spin column hybridization separator system (QIAGEN Inc., Studio City, Calif. followed by oligo(dT) chromatography. The Qiagen-tip was used according to manufacturer's instructions for purification of RNA and run off RNA transcripts.
For either AW108 cRNA or AW109 cRNA, the purified cRNA was quantitated by determining absorbance at 260 nm. The number of molecules present was determined based on the molecular weight of the transcript. AW108 cRNA is 1026 nucleotides in length, therefore, 1 mole=3.386�105 gm (1026�330). Thus, 3.386�105 gm contains 6�1023 cRNA molecules. The number of molecules in 1 pg of AW108 cRNA is (6�1023)/(3.386�105 gm)=1.77�106.
Total cellular RNA was isolated from macrophages and tissues by the method of acid guanidium thiocyanate-phenol-chloroform extraction according to Chomczynski et al., 1987, Analyt. Biochem. 162:156-159.
The cRNA prepared from pAW108 was electrophoresed in 1% low melt agarose, ultra pure grade, in TBE buffer. The region of the gel corresponding to 1 kb was cut out of the gel and melted in 0.2-0.4 ml of 0.1M NETS buffer (0.1M NaCl, 0.01M EDTA; 0.01M Tris-HCl, pH 7.4; 0.2% SDS) containing 1 mM 2-ME, in a water bath at 95� C. for 3-5 minutes and solidified quickly in an ice bucket. The samples were then frozen at -70� C. for at least two hours.
The frozen samples of melted agarose were thawed at 37� C. and centrifuged at top speed in an eppendorf centrifuge kept in the cold room. The agarose was pelleted out. The supernatant liquid was transferred to another eppendorf tube and extracted with a mixture of 100 μl phenol chloroform containing 1% isoamhl alcohol. The phenol was saturated with 0.1M NETS buffer. The aqueous phase was collected, and the RNA was ethanol precipitated. The RNA pellet was washed with 0.1 ml of 2M LiCl and then with 0.1 ml ethanol. The RNA was dried and then dissolved in an appropriate amount of sterile distilled water (2-100 μl) and was ready for reverse transcription.
Oligonucleotides were synthesized on a Biosearch (San Rafael, CA) DNA synthesizer. Most of the primers are RNA-specific primers. The 5' primers spanned the junction of the first two exons and the 3' primers spanned the junction of the next two exons. Alternatively, the 5' primers spanned the junction of the first and second exons and the 3' primers spanned the junction of the second and third exons. These sequences, the genes to which they correspond, and the sizes of amplified products obtained using the primers are shown in Table I.
RNA was reverse transcribed into cDNA as previously described (see Gerard, 1987, Focus (Bethesda Research Labs) 9:5). A 10 μl reverse transcription reaction, containing 1 μg of total cellular RNA, 1.77�102 -1.77�106 molecules of AW108 cRNA, 1�PCR buffer (20 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 100 μg/ml BSA), 1 mM DTT, 0.5 mM dNTP, 10 units RNasin (Promega Biotec), 0.1 μg oligo (dT)12-18, and 100 units of BRL Moloney MuL V reverse transcriptase (Bethesda Research Laboratories) was prepared. The reaction was incubated at 37� C. for 60 minutes, heated to 95� C. for 5-10 minutes, then quickly chilled on ice.
One tenth of the cDNA reaction mixture was diluted in a three-fold dilution series with 0.1 μg/μl tRNA, followed by adjustment to a final concentration of 1�PCR buffer, 50 μM dNTPs, 0.1 μM each of 5' and 3' primers, 1�106 cpm of 32 P end-labeled primer and 1 unit of Taq DNA polymerase (Perkin-Elmer Cetus) in a total volume of 50 μl. The mixture was overlaid with 100 μl mineral oil to prevent evaporation and then amplified for 25 cycles with the Perkin-Elmer Cetus Thermal Cycler. Alternatively, one tenth of the cDNA reaction mixture was amplified using the same conditions as above with varying numbers of cycles. The amplification profile involved denaturation at 95� C. for 30 seconds, primer annealing at 55� C. for 30 seconds, and extension at 72� C. for 1 minute. Oligonucleotides were labeled with γ-32 P-ATP by using polynucleotide kinase and unicorporated nucleotides were removed on a Bio-Gel P-4 column.
Ten μl of each PCR reaction mixture were electrophoresed in 8% polyacrylamide gels in Tris/borate/EDTA buffer. Gels were stained with ethidium bromide and photographed under UV-light irradiation. Appropriate bands were cut from the gel, and radioactivity was determined by Cerenkov counting. The amount of radioactivity recovered from the excised gel bands was plotted against the template concentrations. Data were plotted by exponential curve fitting with a Slide-Write Plus program (Advanced Graphics Software) The amount of target mRNA was quantitated by extrapolating against the AW108 cRNA internal standard curve.
RNA was electrophoresed in a 1.5% agarose gel containing formaldehyde and transferred to a nitrocellulose filter in 20�SSC (1�SCC contained 0.15M sodium chloride and 0.015M sodium citrate). The blot was hybridized with 2�106 cpm of 32 P end-labeled oligonucleotides per ml. Hybridization was for 4 hours at 55� C. in 0.75M NaCl, 0.075M sodium citrate, pH 7.0, 20 mM sodium phosphate, pH 7.0, 5 mM EDTA, 200 μg yeast RNA per ml, and 1% sarkosyl (Sigma). The blot was washed in 1�SSC at 55� C. for 30 minutes and autoradiographed with intensifying screens at -70� C.
Human peripheral blood monocytes were isolated from buffy coat preparations by Ficoll/Hypaque gradient centrifugation followed by adherence to plastic for one hour. Adherent cells were then removed and replated at 106 cells/well onto 6 well plates in RPMI 1640 medium supplemented with 2% fetal calf serum and 2000 units/ml recombinant macrophage-colony stimulating factor (Cetus Corporation). After ten days, half of the cultures were treated with 5 μg/ml LPS (Sigma). All the cultures were harvested for nucleic acid isolation 5 hours later.
The carotid endarterectomy sample was obtained during the course of a surgical operation with the informed consent of the patient. The RNA preparation of a histologically normal coronary artery was recovered from a heart transplant recipient.
As an example of the present method, the AW108 internal standard was used to determine the amount of IL-1α mRNA isolated from LPS-induced cultures of human macrophages. Two different protocols were used to conduct this analysis. In the first case, the amount of template and standard RNAs was varied by serial dilution to generate a standard curve. In the second case, the number of amplification cycles was varied and plotted against the amount of PCR product.
Fifty ng of total macrophage RNA and 1.77�106 molecules of AW108 cRNA were combined and then reverse transcribed into cDNA. Serial three-fold dilutions of one tenth of the cDNA mixture were amplified using the IL-1α specific primers listed in Table 1. About 1�106 cpm of 32 P end-labeled 5' primer were included in the amplification. Reaction products were resolved by gel electrophoresis and visualized by ethidium bromide staining (FIG. 2A). The amounts of radioactivity recovered from the excised gel bands were plotted against the template concentrations (FIG. 2B). In this experiment, target mRNA and AW108 cRNA were amplified after serial three-fold dilutions, and the results demonstrate that the method can resolve less than three-fold differences in RNA concentrations. The fact that the reaction rates of AW108 cRNA and IL-1α mRNA amplification are identical within this exponential phase of the PCR reaction allows construction of a standard curve for AW108 cRNA and extrapolation to a copy number for the IL-1α mRNA present in the macrophages. As shown in FIG. 2B, 1 ng of LPS-induced macrophage total RNA and 1�104 molecules of AW108 cRNA gave the same amount of IL-1α PCR product. In other words, 1 ng of LPS-induced macrophage RNA contained 1�104 molecules of IL-1α mRNA.
Five hundred ng of total macrophage RNA were reverse transcribed with 1.77�106 molecules of AW108 cRNA. Aliquots containing one tenth of the cDNA mixture each were subjected to 14, 16, 18, 20, 22, 24, 26, or 28 cycles of amplification under the same conditions as in protocol E. The amounts of radioactivity recovered from the excised bands were plotted as a function of the number of cycles (FIG. 2C). The rates of amplification were exponential between 14 and 22 cycles for both templates. At 24, 26, and 28 cycles, the rates decreased drastically and approached a plateau. The efficiency of amplification was calculated from the slopes of these curves and found to be 88% for both AW108 cRNA and IL-1α mRNA. Because the amplification efficiency was the same for both co-amplified targets within the exponential phase, the absolute value of IL-1α mRNA can be calculated by comparison with the AW108 cRNA internal standard employing the formula: ##EQU2## where No is the initial amount of material, and N is the extent of amplification. The amount of IL-1α mRNA in 1 ng of LPS-induced macrophage total RNA calculated by this method was 1.1�104 molecules. Thus, the results using either of these two alternative protocols for quantitation are the same.
The amount of IL-1α mRNA in LPS-induced macrophages determined by the quantitative PCR method was verified by Northern blot analysis. The PCR analysis (see above) demonstrated that 1 ng of macrophage RNA and 1�104 molecules of AW108 cRNA produced the same amount IL-1α PCR product. Thus, 5 μg of macrophage RNA and 5�107 molecules of AW108 cRNA should give similar signal intensities by Northern analysis. Two-fold serial dilutions of macrophage RNA and AW108 cRNA were subjected to Northern blot analysis by probing with the IL-1α 3' primer. The sizes of the target RNA molecules were estimated to be ˜2,200 nucleotides for IL-1α mRNA in macrophages and 1026 nucleotides for AW108 cRNA. Hybridization signals of equal intensity were detected at all the dilutions of macrophage RNA and AW108 cRNA, as shown in FIG. 3. This result demonstrates that the amount of mRNA estimated by the quantitative PCR method correlates with the results of Northern analysis.
There are many variables which could influence the effciency of the PCR amplification. Some of the parameters which can be controlled easily are the concentrations of template, dNTPs, MgCl2, primers, polymerase, and PCR cycle profile. However, differences in primer efficiency are difficult parameters to regulate for quantitative analyses. To analyze the primer efficiency effect in the quantitative PCR method, AW108 cRNA was used as the template for PCR amplification of seven different primer sets: IL-1β, PDGF-B, PDGF-B, PDGF-R, IL-2, LPL, and apo-E. As indicated in FIG. 4, the efficiency of amplification by these different primer sets under the same PCR amplification conditions varied over a range of several orders of magnitude. For instance, the IL-1β primers are 105 -fold more efficient than the apo-E primers. Thus, it is critical to use the same primers for amplification of the target mRNA and the internal standard in any attempts to quantitate mRNA expression by PCR.
A major advantage of the present PCR quantitative techniques is that the method enables one to analyze several target mRNA species in parallel. Table III shows the results from quantitation of the expression levels of six cytokine mRNAs in human macrophages in response to LPS treatment. The levels of IL-1β and IL-1α mRNAs, after LPS induction, increased approximately 50-fold. The levels of mRNAs for PDGF-A, M-CSF, and TNF increased 5 to 10-fold. However, the PDGF-B mRNA level remained constant for control and LPS-treated cells. Because the absolute amount of each mRNA was measured, this approach produces a detailed, yet multifaceted picture of the transcriptional phenotype in both the resting and the induced states using only fractions of micrograms of total RNA.
TABLE III______________________________________Specific mRNA levels (molecules/cell)* in LPS-Induced andUninduced Human Macrophages**mRNA Species Uninduced Induced Induced/Uninduced______________________________________IL-1α 1.4 69 49IL-1β 51 2,950 58PDGF-A 0.05 0.48 10PDGF-B 0.47 0.47 1M-CSF 0.06 0.47 8TNF 1.8 8.4 4.7______________________________________ *Molecules/cell = molecules/μg RNA (calculated as in FIG. 2) � μg RNA isolated per cell. **Monocytederived macrophages were cultured for ten days. 5 hours prior t harvest, half of the cultures were exposed to 5 μg/ml LPS.
Because accurate quantitative results can be obtained by the present technology even with small amounts of material, the method is an important tool for the analysis of samples which are rare or in limited quantity, e.g., in vivo-derived biopsy specimens. As an example, Table IV depicts the comparison of the results of quantitation of six different mRNA species from a human, atherosclerotic carotid artery and from a normal coronary artery. The data shows a 3- to 5-fold enhancement in the level of PDGF-A and PDGF-B mRNAs, no change in the type β PDGF receptor (PDGF-R) and a 3-fold decrease in the LDL receptor in the atherosclerotic vessel. There were increases in the levels of IL-1α and IL-1β mRNAs in the diseased tissue.
TABLE IV______________________________________Specific mRNA Levels (Molecules/μg Total RNA)* in aNormal and an Atherosclerotic Blood VesselmRNA Species Atherosclerotic Normal______________________________________PDGF-A 1.8 � 105 3.3 � 104PDGF-B 7.6 � 104 2.2 � 104PDGF-R 1.1 � 104 1.4 � 104LDL-R 4.0 � 103 1.3 � 104IL-1α 1.0 � 102 ND**IL-1β 6.4 � 104 1.0 � 102______________________________________ *Calculated as in FIG. 2. **ND, Not Detectable
Other modifications of the embodiments of the invention described above that are obvious to those of ordinary skill in the areas of molecular biology, medical diagnostic technology, biochemistry, virology, genetics, and related disciplines are intended to be within the scope of the accompanying claims.
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Saturday, March 3, 2012
© Copyright 2015 Gwinnett Daily Post
SUWANEE -- As the meetings and discussions about Suwanee's 20/20 vision reach the home stretch, residents have come with strong opinions, mostly agreements on the city's strengths, and split opinions on only a handful of subjects.
Since June, about 435 residents have been divided into 25 groups to discuss where they want the city to be in eight years. An urban development consultant who the city hired to assist in the project visited this week to give the latest presentation of the findings.
Scott Page, from Interface Studio, made a presentation at Tuesday's City Council meeting and a joint meeting of the facilitator and steering committees on Wednesday. Page said Interface Studio, a Philadelphia-based company, sees its objective as bringing all of the voices together.
The city contracted with Interface Studio for $143,434 to facilitate these discussions over an 11-month timeframe that ends in May. Page's company also is working on similar projects in Wilmington, Del., Detroit and Philadelphia.
The area's residents agree on as strengths of the city were the park system, Town Center and economic development. The most divisive issues were Old Town, public art and the Suwanee Gateway area.
"That's pretty good," Page said. "Usually there's a lot more contention and disagreement. Suwanee is really unique, and that's one of the things we want to emphasize. We want to capitalize about what is distinctive about Suwanee."
Jimmy Spiro, a Suwanee resident, attended Tuesday's council meeting and raised his concern on the lack of discussion about debt reduction, which Page said would be discussed as part of the annual budget process.
"Like any other plan, there's an agenda," Spiro said. "They give you an assignment and an answer to it, and to rank it. Does it give you a vision? Yes. But ... when you came to the very first one, they gave you money, said, 'Here, spend it.' There was not a category to pay down debt."
At Wednesday's meeting, some residents applauded the diversity of their groups; people of all walks of life who have lived in Suwanee for varying lengths of time.
That has given way to input from people of all ages and backgrounds, said resident Dion Jones.
"We have to hang on to some of the great things we have going on here, and try to capitalize on some of the potential the city has moving forward," he said.
Resident Michael Smith said the process has given him a glimpse of the inner workings of the city.
"It's nice to see that on the inside," Smith said. "It's looking at the buildings, and the infrastructure, and bringing the community together, and having a say-so in the future of Suwanee. I think that's what I appreciated about it he most."
Ultimately, the end result will be agreeing to some degree on a list of priorities.
"There will always be varying opinions whenever you bring such a diverse group together," Smith said. "Certainly, there's different motivations that each person brings with them. There will be a lot of spirited debate when it comes to growing a city that has so much character. How do you grow the city, maintain the character and accommodate the growth? That's where most of those friendly debates comes into play."
More like this story
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- 20/20 Vision: Suwanee drafts strategic plan, residents applaud ( May 5, 2012 )