
inn sittu hybridisation

"inn sittu" hybridisation (ishh) ennathu oru tharam hybridisationaanu, athu oru label cheytha pooraka dna, rna allengil parishkkaricha nucleic aasidukal (i.iii.tissuevinte oru bhaagatho vibhaagathilo ("inn sittuvil") allengil tissue vendathra cheruthaanenkil (udha.jii.chedikalude vithukal, "drosophila" bhroonangal), muzhuvan tissuevilum (muzhuvan moundu ishh), koshangalilum, rakthachamkramanam cheiyunna tumour koshangalilum (sii. tii. sii). ithu saadhaaranayaayi tissue vibhagangalil proteinukal praadeshikamaakkunna immunohistolkemisros ninnu vyathyasthamaan.
chromosomukalilo tissuekalilo nirddishta nuclic assid seekwansukalude sthaanam velippeduthaan inn sittu hybridisation upayogikkunnu, ithu jeenukalude oorganaiseshan, niyanthranam, pravartthanam enniva manasilakkunnathinulla nirnaayaka gattamaanu. oligo nucleotide, aarr. enn. eiii probukal (radio-label cheythathum hapton-label cheythathum) upayogichu emm. aarr. enn. eiii. yilekkulla "inn sittu" sankaravalkkaranam, prakashavum electron microscopukalum upayogichu vishakalanam, muzhuvan moundum "inn sittu" sankaravalkkaranam, aarr. enn. eiii, aarr. enn. eiii plus protein ennivayude eratta kandethal, chromosomal sequenceukal kandethunnathinayi fluorescent inn sittu sankaravalkkaranam enniva nilavil upayogikkunna pradhana saankethikavidyakalil ulppedunnu. chromosomukalude ghadana nirnnayikkaan dna ishh upayogikkaam. udaaharanathinu, fluorescent dna ishh (malsyam), chromosomal samagratha vilayiruthunnathin medical diagnosticsil upayogikkaam. tissue vibhagangal, koshangal, muzhuvan moundukal, circulating tumour cellukal (sii. tii. sii) ennivaykkullil aarr. enn. eiii (emm. aarr. eiii, ell. enn. sii. aarr. enn. eiii, mirna) alakkaanum praadeshikavalkkarikkaanum aarr. enn. eiii ishh (aarr. enn. eiii ishh inn sittu) upayogikkunnu. "american jeevashaasthranjaraaya merii-luu pardue, joseph jii. ennivaraanu "inn sittu" sankaravalkkaranam kandupidichathu. gull.
inn-sittu hibridisationte velluvilikal.
tissue vibhaagangalile vyakthigatha kosangalkkullil nirddishta mrna speesheesukal thirichariyunnathinulla sakthamaaya saankethikavidyayaanu inn sittu hybridisation, ithu physiological prakriyakaleyum roga rogakaarikaleyum kurichulla ulkkaazhcha nalkunnu. ennirunnaalum, inn sittu hybridisation parisodhikkappetta oro tissuevinum upayogicha oro probinum krithyamaaya opttimaiseshanodoppam niravadhi nadapadikal kaikkollendathundu. tissukalkkullil taargett mrna samrakshikkunnathinu, cross linking fixetteevukal (formaldehyde polullava) upayogikkendathu aavasyamaanu.
koodaathe, tissue vibhaagangalile inn-sittu sankaravalkkaranathinu tissue sliceukal valare nerthathaayirikkanam, saadhaaranayaayi 3 micrometer muthal 7 micrometer vare kanam. inn-sittu hybridisation prosessinghinaayi tissue vibhagangal thayyarakkunnathinulla saadhaarana reethikalil krayostaatt allengil comprestom tissue slicer upayogichu saambilukal murikkuka ulppedunnu. oru krayostaatt puthiyathum sthiravumaaya tissue eduthu flash freesinginaayi draavaka naitrajanilekku nimajjanam cheiyunnu. thudarnnu tissue oktt ennariyappedunna freeze mediayil ulcherkkukayum nerttha bhagangal murikkukayum cheiyunnu. sariyaaya marnah karaye thadassappeduthunna tissuevil maravippicha karakaushala vasthukkal labhikkunnath thadassangalil ulppedunnu. comprestom oru freeze prakriyayillathe tissue nerttha kashanangalayi murikkunnu; sthirathaykkaayi agarosil ulchertha sesham free-floating vibhagangal murikkunnu. eee reethi tissue maravippikkunnathum athumaayi bandhapetta karakaushala vasthukkal maravippikkunnathum ozhivaakkunnu. eee prakriya poorthiyaayikkazhinjaal sthiravum thirichedukkaanaavaathu.
prakriya.
hybridisation histochemistric, sample cellukalum tissuekalum saadhaaranayaayi taargett traanscriptukal sariyaakkunnathinum probinte axus vardhippikkunnathinum chikilsikkunnu. mukalil soochippichathupole, probe onnukil oru label cheytha pooraka dna allengil ippol ettavum saadhaaranayaayi oru pooraka rna (riboprobe) aanu. uyarnna thapanilayil probe taargett seekwansilekku sankaravalkarikkappedukayum thudarnnu adhika probe kazhuki kalayukayum cheiyunnu (unhybridised, adhika rna probinte kaaryatthil arnez upayogichu munkaala jalavishleshanathinu sesham). thaapanila, uppu, koodaathe/allengil dittarjant saandrata thudangiya laayaniyude paaraamettarukal samaanamallatha edapedalukal neekkam cheiyunnathinu kaikaaryam cheyyaan kazhiyum (i.iii.krithyamaaya sequence malsarangal maathrame sheshikkunnulloo). pinne, radio-, fluorescent-allengil aantijan-label cheytha bays (udha.jii.degoxyjenin) yathaakramam autoradiography, fluorescence microscopy allengil immunohistokemisro enniva upayogichu tissuevil praadeshikavalkkarikkuppudu alakkukayum cheiyunnu. rando athiladhikamo traanscriptukal oresamayam kandethunnathinu radio aaktivitti allengil mattu noonn-radio active labelukal upayogichu label cheytha rando athiladhikamo probukal upayogikkaanum aii. ezz. echu.-nuu kazhiyum.
radio aaktivitti upayogikkaathe orotta thanmatra samvedanakshamathayulla rna (mrna, elncrna, mirna) "inn sittu" hybridisation parishodhanakalkk branched dna asse enna badal saankethikavidya upayogikkaam. eee sameepanam (udha.jii.viewerna assaisukal) oru aseyil naalu targettukal vare drishyavalkkarikkaan upayogikkaam, koodaathe ithu pettant cheytha probe design bdna signal amplification upayogichu sensiteevum nirddishtavumaaya signalukal srishtikkunnu. saambilukal (koshangal, tissukal, sii. tii. sii. kall) sthirappeduthukayum thudarnnu aarr. enn. eiii taargett accessibility (aarr. enn. eiii ann-masking) anuvadikkunnathinu chikilsikkukayum cheiyunnu. taargett-nirddishta probukal oro taargett arnailekkum sankaravalkkarikkunnu. aduthulla probukalude (aarr. enn. eiii. lakshyangalil arikil bandhippikkunna vyakthigatha oligo nucleotideukal [oligos]) nirddishta sankaravalkkaranathe adisthaanamaakkiyaanu thudarnnulla signal amplification pravachikkunnath. oru saadhaarana taargett-nirddishta probil 40 oligo nucleotideukal adangiyirikkum, athinte falamaayi 20 oligo jodikal mrna, elncra enniva kandethunnathinayi lakshyathil arikil bandhippikkunnu, koodaathe 2 oligos allengil mirna kandethunnathinayi orotta jody. thudarchayaaya sankaravalkkarana ghattangaliloode signal amplification nedaam. oru pree-amplifier thanmatra taargett-nirddishta rnaile oro oligo jodiyilekkum sankaravalkkarikkappedunnu, thudarnnu onniladhikam amplifier thanmathrakal oro pree-amplifierilekkum sankaravalkkarikkappedunnu. aduthathaayi, onniladhikam label probe oligo nucleotideukal (alkaline phosphatasilekko nerittu fluoroforukalilekko samyojippichirikkunnu) oro amplifier thanmathrayilekkum sankaravalkkarikkappedunnu. poornnamaayum kootticherttha signal amplification gadanayaaya "trea" yill label probukalkkaayi 400 binding saittukalund. ellah taargett-nirddishta probukalum taargett mrna traanscriptumaayi bandhippikkumbol, aaa oru traanscriptinu 8,000 madangu signal amplification sambhavikkunnu. vyathyasthavum ennaal anuyojyavumaaya signal amplification samvidaanangal multiplexes parishodhanakale prapthamaakkunnu. fluorescence allengil braitfield microscope upayogichu signal drishyavalkkarikkaam.
degoxyjenin-label cheytha probukalkkulla adisthaana gattangal.
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