I-RT-qPCR luvavanyo olusisiseko lwebhayoloji yemolekyuli, kwaye wonke umntu kufuneka aqhelane nayo.Ibandakanya ikakhulu amanyathelo amathathu: ukutsalwa kwe-RNA, ukukhutshelwa umva kwi-cDNA, kunye nexesha lokwenyani le-fluorescent quantitative PCR.Akuncedi, kuqhubeka ntoni?Kusenokwenzeka ukuba kukho ingxakiumfuniselo wokukhutshelwa umva!Nangona kubonakala ngathi umfuniselo wokukhutshelwa umva ufuna kuphela ukongeza i-RNA, i-dNTP, iiprimers, kunyereverse transcriptasekwityhubhu ye-centrifuge kwaye udibanise kakuhle, kodwa kwinkqubo yokusebenza yangempela, kusekho iinkcukacha ezininzi ezifuna ukuhoywa.Masifunde ngayo!

Indlela yokugweba umgangatho we-RNA?
Ukufumana i-cDNA, umgangatho we-RNA ubalulekile!Umgangatho we-RNA unokubonwa ikakhulu kwimiba emibini:
(1) Ingqibelelo yeRNA:Ukunyaniseka kwe-RNA kunokuqinisekiswa nge-agarose gel electrophoresis. Ukuthatha i-eukaryotes njengomzekelo, i-RNA epheleleyo epheleleyo inamaqela amathathu acacileyo, ubunzima be-molecular ukusuka kubukhulu ukuya kuncinci yi-28S, i-18S, kunye ne-5S, kunye ne-28S iphindwe kabini njenge-18S;ukuba amaqela amathathu angabonwa, kodwa uhlobo lwebhendi lumfiliba okanye Ukusasazwa kuthetha ukuba i-RNA ithotywe ngokuyinxenye.Ngeli xesha, nceda wenze ireverse transcription reaction ngoko nangoko kwaye wandise igalelo letemplate ngokufanelekileyo;ukuba kuphela ibhanti enobunzima obuncinane bemolekyuli okanye akukho bhanti inokubonwa, iRNA iye yathotywa ngokupheleleyo yaye kufuneka iphinde ikhutshwe .I-Agilent 2100 ibonisa ukunyaniseka kwe-RNA kunye nomzobo ophakamileyo kunye nexabiso le-RIN.Ukuba i-nucleic acid ilungile, isiseko se-electropherogram sicaba;ukuba i-asidi ye-nucleic ihlaziywa kakhulu, isiseko asilingani kwaye iincopho zokunciphisa zivela;ixabiso le-RIN libonisa ukunyaniseka kwe-RNA, ngaphakathi koluhlu lwe-0-10, ixabiso elikhulu, elingcono umgangatho we-RNA.Ewe, inqanaba eliphezulu lokugqibelela.
(2) Ubunyulu be-RNA:Umlinganiselo we-OD260/280 unokubonwa nge-UV spectrophotometry.Ukuba umlinganiselo we-OD260/280 uphakathi kwe-1.9 kunye ne-2.1, ukucoceka kuhle kakhulu.
I-DNA eseleyo ye-genomic inokukhokelela kwiziphumo ezingachanekanga zobungakanani
Xa i-RNA ikhutshwa, i-RNA esiyifumanayo inokuxutywa ne-genomic DNA (gDNA) engazange icocwe.Ke ngoko, i-cDNA emva kokukhuphela umva iyakuxutywa nayogDNA.Ngexesha lomlambo osezantsiqPCRimpendulo,cDNAkunye ne-gDNA inokwandiswa ngaxeshanye, okukhokelela kwixabiso elincinci le-CT, ngoko ke iziphumo zinokuthi zibe nomkhethe.
Ngoko sifanele senze ntoni kule meko?I-Foregeneicebisa:
(1) Yenza ukucoca i-genome kwi-RNA eguqulwayo, enokuthi isuswe nge-column extraction ngexesha lokukhutshwa kwe-RNA;
(2) Phatha i-RNA ekhutshwe nge-DNaseI , kodwa yiphelise nge-EDTA;
yereverse transcription reagentngeemodyuli zokucoca i-genome;

Ungakhetha njani iiprimers zokukhuphela umva?
Reverse transcription primers ikwachaphazela isiphumo sereverse transcription reaction.Unokukhetha iiprimers ezingahleliweyo, i-Oligo dT okanye i-gene-specific primers yokukhuphela umva ngokweemeko ezithile zovavanyo:
(1) Imibhalo ethile: iiprimers zegene-specific ziyacetyiswa;
(2) Iziqwenga ezide ezikhutshelweyo: I-Oligo dT/i-gene-specific primers iyacetyiswa;
(3) Iziqwenga zangaphakathi zemibhalo yecandelo elide: ii-primers ze-gene-specific/ ii-primers ezingaqhelekanga / ii-primers ezingaqhelekanga + i-Oligo dT.Ukuba uvavanyo olulandelayo lwe-qPCR luyenziwa, i-Oligo dT ayinakusetyenziswa yodwa, kuba ukusebenzisa i-Oligo dT yodwa kunokubangela ukuba i-3′ iphele i-bias, ekhokelela kwiziphumo zokulinga ze-qPCR ezingachanekanga;
(4) miRNA: Ii-primers ze-stem-loop okanye ii-primers ze-tailing zingasetyenziswa.

Mangaphi amaxesha ekufuneka imveliso yokukhuphela umva i-cDNA ihlanjululwe kumlinganiselo?
Emva kokufumana i-cDNA yemveliso ekhutshelweyo ebuyela umva, mangaphi amaxesha ekufuneka i-cDNA ixutywe kuvavanyo lwe-qPCR ibaluleke kakhulu.Ukuba i-cDNA yoxinaniso iphezulu kakhulu okanye iphantsi kakhulu, ukusebenza kakuhle kokukhulisa kunokuchaphazeleka.Ngaba i-cDNA yoxinaniso inokulinganiswa, kwaye kufuneka yenziwe njani?
(1) Uxinaniso lwe-cDNA yemveliso yokukhuphela i-reverse ayinakulinganiswa, kuba ngaphezu kwemveliso ye-cDNA, imveliso ye-reverse transcription nayo iqulethe i-reverse transcription residual Buffer, i-reverse transcriptase, i-primers, njl., eya kuphazamisa iziphumo zokulinganisa ukugxininiswa kwaye ibangele i-OD260 / 280, i-OD260 / 230 imveliso ye-cDNA ayibonakalisi.Ngeli xesha, abanye abahlobo baya kuthi, ngoko ndiya kulinganisa ugxininiso emva kokuhlanjululwa;Apha, iForgene ingathanda ukukhumbuza ukuba i-cDNA ayikhuthazwa ukuba ihlanjululwe, kuba ubude be-cDNA efunyenwe ngokuguqulwa buhluke, kwaye i-cDNA emfutshane iya kulahleka ekuhlanjululeni.
(2) Masenze ntoni ke?Phambi kovavanyo lwe-qPCR, i-dilution gradient ye-cDNA inokumiselwa ngovavanyo lwangaphambili.Umzekelo: sebenzisa isisombululo sesitokhwe se-cDNA, i-10-fold dilution, kunye ne-100-fold dilution njengeetemplates zovavanyo lwe-qPCR, kwaye ukhethe i-dilution factor enexabiso le-CT kuluhlu lwe-18-28.

Ii-miRNA kufuneka zikhutshelwe njani umva?
i-miRNA yimolekyuli encinci enemisonto enye ye-RNA enobungakanani obumalunga ne-22 nt engayifaki ikhowudi yeprotheyini.Ngenxa yobude bayo obufutshane, indlela yesiqhelo ye-qPCR kunzima ukuyilinganisa ngokuthe ngqo, ngoko kuye kufuneke ukuba kwandiswe i-miRNA;iindlela ezixhaphakileyo zokukhuphela umva we-miRNA ziquka indlela ye-stem-loop kunye nendlela yomsila.
Indlela ye-stem-loop kukwandisa i-miRNA ngokudibanisa i-stem-loop primers.Le ndlela yokufumanisa inovakalelo oluphezulu kunye neenkcukacha ezithile, kodwa ukubonwa kwe-output kuphantsi.Ushicilelo olunye olubuyela umva luyakwazi ukubona i-miRNA enye kunye nesalathiso sangaphakathi;indlela yokongeza umsila iqulethwe ezimbini Igqitywe ngesenzo esidibeneyo se-enzymes ezimbini, eziyi-PolyA polymerase kunye ne-reverse transcriptase.I-PolyA polymerase inoxanduva lokongeza i-PolyA imisila kwi-miRNA ukwandisa ubude bayo, kwaye i-reverse transcriptase yenza i-reverse transcript reaction.Le ndlela inokubonwa okuphezulu kwaye iyakwazi ukubona ii-miRNA ezininzi kunye neereferensi zangaphakathi kwi-reverse transcription, kodwa uvakalelo kunye nokuchaneka kuphantsi kwindlela ye-stem-loop.