I-RT-qPCR ukuhlola okuyisisekelo kwebhayoloji yamangqamuzana, futhi wonke umuntu kufanele ayazi.Ihlanganisa ikakhulukazi izinyathelo ezintathu: Ukukhishwa kwe-RNA, ukuloba okuhlanekezelwe ku-cDNA, kanye ne-PCR yesikhathi sangempela ye-fluorescent quantitative.Akusizi, kwenzakalani?Kungenzeka ukuthi kunenkinga ngeisilingo sokuhlehla sokuloba!Nakuba kubonakala sengathi ukuhlolwa kokulotshiweyo okuhlanekezelwe kudinga kuphela ukwengeza i-RNA, i-dNTP, iziqalo, kanyereverse transcriptaseku-tube ye-centrifuge bese uxuba kahle, kodwa ekusebenzeni kwangempela, kusenemininingwane eminingi okudingeka inakwe.Ake sifunde ngakho!

Indlela yokwahlulela ikhwalithi ye-RNA?
Ukuthola i-cDNA, ikhwalithi ye-RNA ibalulekile!Ikhwalithi ye-RNA ingatholwa ikakhulukazi ezicini ezimbili:
(1) Ubuqotho be-RNA:Ubuqotho be-RNA bungaqinisekiswa yi-agarose gel electrophoresis. Uma sithatha ama-eukaryote njengesibonelo, isamba esiphelele se-RNA inamabhande amathathu acacile, izisindo zamangqamuzana kusukela kwezinkulu kuya ezincane zingu-28S, 18S, no-5S, kanti u-28S ukhanya ngokuphindwe kabili njengo-18S;uma amabhendi amathathu engabonwa, kodwa uhlobo lwebhendi lufiphalisiwe noma Ukusabalalisa kusho ukuthi i-RNA yehliswe kancane.Ngalesi sikhathi, sicela wenze ukusabela kokuhlehla kokulotshiweyo ngokushesha futhi wandise okokufaka kwesifanekiso ngokufanele;uma kuphela ibhande elinesisindo esincane samangqamuzana noma lingekho ibhande elingabonwa, i-RNA yehliswe ngokuphelele futhi idinga ukukhishwa kabusha .I-Agilent 2100 ibonisa ubuqotho be-RNA enomdwebo ophakeme kanye nenani le-RIN.Uma i-nucleic acid iqinile, isisekelo se-electropherogram siyisicaba;uma i-nucleic acid yonakaliswa kakhulu, isisekelo asilingani futhi kuvela iziqongo zokuwohloka okwengeziwe;inani le-RIN libonisa ubuqotho be-RNA, ngaphakathi kwebanga lika-0-10, inani elikhulu, ikhwalithi ye-RNA engcono.Hhayi-ke, izinga lokuphelela liphakeme.
(2) Ukuhlanzeka kwe-RNA:Isilinganiso se-OD260/280 singatholwa nge-UV spectrophotometry.Uma isilinganiso se-OD260/280 siphakathi kuka-1.9 no-2.1, ubumsulwa buhle kakhulu.
I-DNA eyinsalela ye-genomic ingaholela emiphumeleni yobuningi obungalungile
Uma i-RNA ikhishwa, i-RNA esiyitholayo ingase ixutshwe ne-genomic DNA (gDNA) engakahlanzwa.Ngakho-ke, i-cDNA ngemva kokuloba okuhlanekezelwe nayo izoxutshwa nayogDNA.Ngesikhathi somfula ongezansiqPCRukusabela,cDNAfuthi i-gDNA ingase ikhuliswe ngesikhathi esisodwa, okuholela enanini elincane le-CT, ngakho imiphumela ingase icheme.
Ngakho yini okufanele siyenze kulesi simo?I-Foregeneiphakamisa:
(1) Yenza ukuhlanza i-genome ku-RNA ehlehlisiwe, engasuswa ngokukhipha ikholomu phakathi nokukhipha i-RNA;
(2) Phatha i-RNA ekhishiwe nge-DNaseI , kodwa iqede nge-EDTA;
yama-reverse transcription reagentngamamojula wokuhlanza i-genome;

Ungawakhetha kanjani ama-primers wokulotshwa okuhlanekezelwe?
Iziqalo zokuloba ezihlehliswayo nazo zithinta umphumela wokusabela kokuhlehla kokulotshiweyo.Ungakhetha ama-primer angahleliwe, i-Oligo dT noma ama-primer aqondene nofuzo ukuze ulotshwe ngokuhlehla ngokuya ngezimo ezithile zokuhlolwa:
(1) Imibhalo ethize: Iziqalo eziqondene nofuzo ziyanconywa;
(2) Okulotshiweyo kweziqephu ezinde: Kunconywa ama-primers we-Oligo dT/gene-specific;
(3) Izingcezu zangaphakathi zemibhalo yezingxenye ezinde: ama-primer aqondene nofuzo oluthize/ ama-primer angahleliwe /okuqala okungahleliwe + Oligo dT.Uma ukuhlolwa okulandelayo kwe-qPCR kwenziwa, i-Oligo dT ayikwazi ukusetshenziswa iyodwa, ngoba ukusebenzisa i-Oligo dT iyodwa kungase kubangele ukuchema kokuphela kwe-3, okuholela emiphumeleni yokuhlola ye-qPCR engalungile;
(4) i-miRNA: Iziqalo ze-stem-loop noma iziqalo zomsila zingasetshenziswa.

Kufanele uhlanjululwe kangaki umkhiqizo wombhalo ohlanekezelwe we-cDNA ukuze ulinganiswe?
Ngemva kokuthola i-cDNA yomkhiqizo wokulotshwa okuhlanekezelwe, ukuthi i-cDNA kufanele ihlanjululwe kangaki ekuhloleni kwe-qPCR kubaluleke kakhulu.Uma ukugxila kwe-cDNA kuphezulu kakhulu noma kuphansi kakhulu, ukusebenza kahle kokukhulisa kungase kuthinteke.Ingabe ukugxila kwe-cDNA kungalinganiswa, futhi kufanele kwenziwe kanjani?
(1) Ukugxiliswa kwe-cDNA komkhiqizo wokulotshwa okuhlanekezelwe akukwazi ukulinganiswa, ngenxa yokuthi ngaphezu komkhiqizo we-cDNA, umkhiqizo wokulotshwa okuhlanekezela futhi uqukethe i-Reverse transcriptase residual Buffer, reverse transcriptase, primers, njll., okuzophazamisa imiphumela yesilinganiso sokuhlushwa futhi kubangele i-OD260/280, OD260/ 230 ngakho-ke isilinganiso se-cDNA asibonisi iqiniso.Ngalesi sikhathi, abanye abangani bazothi, khona-ke ngizokala ukugxilisa ingqondo ngemva kokucwengwa;lapha, uForgene uthanda ukukhumbuza ukuthi i-cDNA ayinconywa ukuba ihlanzwe, ngoba ubude be-cDNA etholwe ngokuhlehliswa buhlukile, futhi i-cDNA emfushane izolahleka ekuhlanzweni.
(2) Pho yini okumele uyenze?Ngaphambi kokuhlolwa kwe-qPCR, i-dilution gradient ye-cDNA inganqunywa ngokuhlolwa kwangaphambilini .Isibonelo: sebenzisa isixazululo sesitoko se-cDNA, i-dilution ephindwe ka-10, kanye ne-dilution ephindwe ka-100 njengezifanekiso zokuhlolwa kwe-qPCR, bese ukhetha isici sokuhlanjululwa ngevelu ye-CT ebangeni lika-18-28.

Ama-miRNA kufanele abhalwe kanjani emuva?
I-miRNA iyi-molecule encane enomucu owodwa we-RNA enosayizi ongaba ngu-22 nt ongawakhiphi amakhodi amaprotheni.Ngenxa yobude bayo obufushane, indlela evamile ye-qPCR kunzima ukuyilinganisa ngokuqondile, ngakho-ke kuvame ukudingekile ukunweba i-miRNA;izindlela ezivame ukusetshenziswa zokuhlehla zokuloba ze-miRNA zifaka indlela ye-stem-loop kanye nendlela yokusile.
Indlela ye-stem-loop iwukunweba i-miRNA ngokungeza iziqalo ze-stem-loop.Le ndlela yokuthola inokuzwela okuphezulu nokucaciswa kwayo, kodwa ukutholwa kwe-throughput kuphansi.Okulotshiweyo okukodwa okuphambene kungathola kuphela i-miRNA eyodwa kanye nereferensi yangaphakathi;indlela yokwengeza umsila yakhiwe ngamabili Iqedwa isenzo esihlangene sama-enzyme amabili, okuyi-PolyA polymerase kanye ne-reverse transcriptase.I-PolyA polymerase inesibopho sokwengeza umsila we-PolyA ku-miRNA ukuze ikhulise ubude bayo, futhi i-reverse transcriptase yenza ukusabela kokuhlehla kokuloba.Le ndlela inokutholwa okuphezulu futhi ingathola ama-miRNA amaningi nezithenjwa zangaphakathi ekulotshweni okuphambene okukodwa, kodwa ukuzwela nokucaciswa kuphansi kundlela ye-stem-loop.