RT-qPCR ndiye kuyesa koyambirira kwa biology ya maselo, ndipo aliyense ayenera kuidziwa bwino.Zimaphatikizanso masitepe atatu: kutulutsa kwa RNA, kusindikizanso ku cDNA, ndi PCR yeniyeni ya fulorosenti.Sizikuthandizira, chikuchitika ndi chiyani?Zikuoneka kuti pali vuto ndikuyesa kumasulira mobwereza!Ngakhale zikuwoneka kuti kuyesa kobwerezabwereza kumangofunika kuwonjezera RNA, dNTP, zoyambira, ndireverse transcriptaseku chubu cha centrifuge ndikusakaniza bwino, koma muzochitika zenizeni, pali zambiri zomwe ziyenera kutsatiridwa.Tiyeni tiphunzire za izo!

Momwe mungadziwire mtundu wa RNA?
Kuti mupeze cDNA, mtundu wa RNA ndiofunikira!Makhalidwe a RNA amatha kudziwika makamaka kuchokera kuzinthu ziwiri:
(1) Kukhulupirika kwa RNA:Umphumphu wa RNA ukhoza kutsimikiziridwa ndi agarose gel electrophoresis. Kutengera chitsanzo cha eukaryotes, RNA yonse ili ndi magulu atatu omveka bwino, zolemera za maselo kuchokera ku zazikulu mpaka zazing'ono ndi 28S, 18S, ndi 5S, ndipo 28S ndi yowala kawiri kuposa 18S;ngati magulu atatu atha kuwoneka, koma mtundu wa gulu ndi wodetsedwa kapena Diffusion zikutanthauza kuti RNA yawonongeka pang'ono.Pakadali pano, chonde chitani zolembera mobwerera m'mbuyo nthawi yomweyo ndikuwonjezera zolowetsamo moyenera;ngati gulu lokhala ndi cholemetsa chaching'ono cha mamolekyu kapena palibe gulu lomwe lingawonekere, RNA yawonongeka kotheratu ndipo iyenera kuchotsedwanso.Agilent 2100 ikuwonetsa kukhulupirika kwa RNA yokhala ndi chithunzi chapamwamba komanso mtengo wa RIN.Ngati nucleic acid ili bwino, maziko a electropherogram ndi athyathyathya;ngati nucleic acid yawonongeka kwambiri, maziko ake ndi osagwirizana ndipo nsonga zowonongeka zimawonekera;mtengo wa RIN umasonyeza kukhulupirika kwa RNA, mkati mwa 0-10, mtengo wokulirapo, ubwino wa RNA.Chabwino, ndipamwamba mlingo wa kukwanira.
(2) Kuyera kwa RNA:Chiŵerengero cha OD260/280 chikhoza kudziwika ndi UV spectrophotometry.Ngati chiŵerengero cha OD260/280 chiri pakati pa 1.9 ndi 2.1, chiyero ndi chabwino kwambiri.
DNA yotsalira ya genomic imatha kubweretsa zotsatira zolakwika za kuchuluka
RNA ikatulutsidwa, RNA yomwe timapeza imatha kusakanikirana ndi genomic DNA (gDNA) yomwe sinayeretsedwe.Chifukwa chake, cDNA pambuyo polemba m'mbuyo idzasakanizidwansogDNA.M'nyengo yoziziraqPCRzomwe,cDNAndipo gDNA ikhoza kukulitsidwa nthawi imodzi, zomwe zimapangitsa kuti pakhale mtengo wochepa wa CT, kotero zotsatira zake zingakhale zokondera.
Ndiye tiyenera kuchita chiyani pamenepa?Foregeneamalimbikitsa:
(1) Chitani matupi athu kuyeretsa pa RNA zosinthidwa, amene akhoza kuchotsedwa ndi m'zigawo m'zigawo pa RNA m'zigawo;
(2) Chitani RNA yotengedwa ndi DNaseI , koma kuithetsa ndi EDTA;
za reverse transcription reagentsndi ma modules ochotsa ma genome;

Momwe mungasankhire zoyambira za reverse transcription?
Zoyambira zomasulira zobwerera m'mbuyo zimakhudzanso zotsatira za kalembedwe ka m'mbuyo.Mutha kusankha zoyambira mwachisawawa, Oligo dT kapena zoyambira zenizeni zamtundu kuti mulembe mobwerera molingana ndi zomwe zayesererazo:
(1) Zolemba zenizeni: zoyambira za jini zimalimbikitsidwa;
(2) Zolemba zazitali zachidutswa: Oligo dT/ma jini oyambira akulimbikitsidwa;
(3) Zidutswa zamkati za zolemba zazitali: zoyambira za jini / zoyambira mwachisawawa / zoyambira mwachisawawa + Oligo dT.Ngati kuyesa kotsatira kwa qPCR kuchitidwa, Oligo dT sangathe kugwiritsidwa ntchito yekha, chifukwa kugwiritsa ntchito Oligo dT yekha kungayambitse 3 'kukondera komaliza , zomwe zimapangitsa zotsatira zolakwika za qPCR;
(4) miRNA: Zoyambira za tsinde kapena zoyambira zoyambira zitha kugwiritsidwa ntchito.

Kodi cDNA iyenera kuchepetsedwa kangati kuti quantification?
Mutapeza cDNA ya zinthu zolembedwa m'mbuyo, kangati cDNA iyenera kuchepetsedwa pazoyeserera za qPCR ndikofunikira kwambiri.Ngati ndende ya cDNA ndiyokwera kwambiri kapena yotsika kwambiri, mphamvu yokulitsa imatha kukhudzidwa.Kodi kuchuluka kwa cDNA kungayesedwe, ndipo kuyenera kuchitika bwanji?
(1) Kuchulukira kwa cDNA kwa zinthu zolembedwa m'mbuyo sikungayesedwe, chifukwa kuwonjezera pa chinthu cha cDNA, chosindikizira cha reverse transcript chimakhalanso ndi reverse transcript residual Buffer, reverse transcriptase, primers, etc., zomwe zingasokoneze zotsatira za kuyeza kwa ndende ndikuyambitsa OD260/280, OD260/ 230 chiŵerengero cha cDNA sichisonyeza.Panthawiyi, abwenzi ena adzati, ndiye ndidzayesa ndende pambuyo pa kuyeretsedwa;apa, Forgene akufuna kukumbutsa kuti cDNA sivomerezedwa kuti iyeretsedwe, chifukwa utali wa cDNA wopezedwa ndi kusinthika ndi wosiyana, ndipo cDNA yaifupi idzatayika pakuyeretsedwa.
(2) Ndiye titani?Kuyesa kwa qPCR kusanachitike, kukwera kwa dilution kwa cDNA kumatha kudziwitsidwa kudzera mu kuyesa koyambirira.Mwachitsanzo: gwiritsani ntchito cDNA stock solution, 10-fold dilution, and 100-fold dilution as templates for qPCR experiments, and sankhani dilution factor yokhala ndi CT value of 18-28.

Kodi ma miRNA ayenera kulembedwa bwanji m'mbuyo?
miRNA ndi molekyu yaing'ono yokhala ndi chingwe chimodzi ya RNA yokhala ndi kukula pafupifupi 22 nt yomwe ilibe puloteni.Chifukwa chautali wake waufupi, njira yodziwika bwino ya qPCR ndiyovuta kuwerengera mwachindunji, kotero nthawi zambiri ndikofunikira kuwonjezera miRNA;Njira zomwe zimagwiritsidwa ntchito mobwerezabwereza za miRNA zikuphatikizapo njira ya tsinde-loop ndi njira ya tailing.
Njira ya stem-loop ndikukulitsa miRNA powonjezera zoyambira za stem-loop.Njira yodziwikirayi ili ndi chidwi chambiri komanso mwachindunji, koma njira yodziwira ndiyotsika.Kusindikiza kumodzi kutha kuzindikira miRNA imodzi ndi kalozera wamkati;njira yowonjezera mchira imapangidwa ndi awiri Imatsirizidwa ndi mgwirizano wa michere iwiri, yomwe ndi PolyA polymerase ndi reverse transcriptase.PolyA polymerase ndiyomwe ili ndi udindo wowonjezera michira ya PolyA ku miRNA kuti ionjezere utali wake, ndipo reverse transcriptase imachita reverse transcript reaction.Njirayi imakhala ndi njira yodziwira kwambiri ndipo imatha kuzindikira ma miRNA angapo ndi maumboni amkati muzolemba zotsatizana, koma kukhudzidwa ndi kutsimikizika ndizochepa mu njira ya tsinde-loop.