RT-qPCR yog qhov kev sim yooj yim ntawm molecular biology, thiab txhua tus yuav tsum paub txog nws.Nws feem ntau suav nrog peb kauj ruam: RNA rho tawm, thim rov qab rau hauv cDNA, thiab lub sijhawm fluorescent quantitative PCR.Nws tsis pab, dab tsi tshwm sim?Nws zoo li muaj teeb meem nrogqhov kev sim rov qab transcription!Txawm hais tias nws zoo li qhov kev sim rov qab hloov pauv tsuas yog xav tau ntxiv RNA, dNTP, primers, thiabrov qab transcriptasemus rau lub centrifuge raj thiab sib tov zoo, tab sis nyob rau hauv lub sij hawm ua hauj lwm txheej txheem, tseem muaj ntau yam ntsiab lus uas yuav tsum tau them sai sai rau.Cia peb kawm txog nws!

Yuav txiav txim qhov zoo ntawm RNA li cas?
Yuav kom tau txais cDNA, qhov zoo ntawm RNA yog qhov tseem ceeb!RNA zoo tuaj yeem kuaj pom los ntawm ob yam:
(1) RNA kev ncaj ncees:RNA kev ncaj ncees tuaj yeem txheeb xyuas los ntawm agarose gel electrophoresis. Noj eukaryotes ua piv txwv, tag nrho RNA muaj peb qhov tseeb bands, qhov hnyav molecular los ntawm loj mus rau me me yog 28S, 18S, thiab 5S, thiab 28S yog ob zaug li ci li 18S;yog peb pawg tuaj yeem pom, tab sis hom band yog qhov muag plooj lossis Diffusion txhais tau hais tias RNA yog ib feem ntawm kev puas tsuaj.Nyob rau lub sijhawm no, thov ua qhov kev hloov pauv rov qab tam sim ntawd thiab ua kom cov qauv kev nkag siab tsim nyog;Yog tias tsuas yog ib pawg me me me me los sis tsis muaj cov qhab tuaj yeem pom, RNA tau raug rhuav tshem tag nrho thiab yuav tsum tau rov muab rho tawm.Agilent 2100 qhia txog kev ncaj ncees ntawm RNA nrog lub ncov daim duab thiab RIN tus nqi.Yog tias cov nucleic acid tsis zoo, lub hauv paus ntawm electropherogram yog tiaj;yog hais tias cov nucleic acid yog degraded heev, lub hauv paus yog tsis sib xws thiab ntau degradation peaks tshwm;Tus nqi ntawm RIN qhia txog kev ncaj ncees ntawm RNA, nyob rau hauv thaj tsam ntawm 0-10, qhov loj dua tus nqi, qhov zoo ntawm RNA.Zoo, qhov siab dua qhov ua tiav.
(2) Purity ntawm RNA:Qhov piv ntawm OD260/280 tuaj yeem kuaj pom los ntawm UV spectrophotometric .Yog tias qhov piv ntawm OD260/280 yog nyob nruab nrab ntawm 1.9 thiab 2.1, qhov purity yog qhov zoo heev.
Residual genomic DNA tuaj yeem ua rau cov txiaj ntsig tsis raug
Thaum RNA raug rho tawm, RNA peb tau txais yuav raug muab sib xyaw nrog genomic DNA (gDNA) uas tsis tau raug ntxuav.Yog li ntawd, cov cDNA tom qab kev hloov pauv rov qab kuj tseem yuav sib xyaw nroggDNA ua.Thaum lub caij nplooj zeegqPCRtshuaj tiv thaiv,cDNAthiab gDNA tuaj yeem nthuav dav ib txhij, ua rau muaj tus nqi CT me me, yog li cov txiaj ntsig tuaj yeem ua tsis ncaj.
Yog li peb yuav tsum ua li cas hauv qhov xwm txheej no?Foregeneqhia:
(1) Ua cov genome tu ntawm qhov thim rov qab RNA, uas tuaj yeem raug tshem tawm los ntawm kem rho tawm thaum RNA rho tawm;
(2) Kho RNA rho tawm nrog DNaseI , tab sis txiav nws nrog EDTA;
ntawm reverse transcription reagentsnrog genome clearing modules;

Yuav ua li cas xaiv primers rau thim rov qab?
Reverse transcription primers kuj cuam tshuam qhov tshwm sim ntawm qhov rov qab transcription cov tshuaj tiv thaiv.Koj tuaj yeem xaiv random primers, Oligo dT lossis gene-specific primers rau thim rov qab raws li cov xwm txheej tshwj xeeb ntawm kev sim:
(1) Cov ntawv sau tshwj xeeb: gene-specific primers raug pom zoo;
(2) Cov ntawv teev lus ntev: Oligo dT/gene-specific primers raug pom zoo;
(3) Internal fragments ntawm ntu ntev transcripts: gene-specific primers / random primers / random primers + Oligo dT.Yog tias qhov kev sim qPCR tom qab tau ua tiav, Oligo dT tsis tuaj yeem siv ib leeg, vim tias siv Oligo dT ib leeg tuaj yeem ua rau 3′ kawg kev tsis ncaj ncees, ua rau tsis raug qPCR cov txiaj ntsig kev sim;
(4) miRNA: Stem-loop primers lossis tailing primers tuaj yeem siv tau.

Yuav ua li cas muaj pes tsawg lub sij hawm yuav tsum rov qab transcription khoom cDNA diluted rau quantification?
Tom qab tau txais cDNA ntawm cov khoom siv rov qab, yuav tsum muaj pes tsawg zaus cDNA yuav tsum tau diluted rau qPCR kev sim yog qhov tseem ceeb heev.Yog tias cDNA concentration siab dhau los yog qis dhau, qhov kev ua kom zoo dua tuaj yeem cuam tshuam.Puas tuaj yeem ntsuas cDNA concentration, thiab nws yuav tsum ua li cas?
(1) Lub cDNA concentration ntawm cov khoom rov qab transcription tsis tuaj yeem ntsuas, vim hais tias ntxiv rau cov khoom cDNA, cov khoom rov qab rov qab kuj tseem muaj cov ntaub ntawv rov qab uas tsis muaj, rov qab transcriptase, primers, thiab lwm yam, uas yuav cuam tshuam nrog kev ntsuas qhov ntsuas thiab ua rau OD260/280, OD260 / 230 tsis muaj tseeb thiab qhov sib piv ntawm CNA.Lub sijhawm no, qee cov phooj ywg yuav hais tias, tom qab ntawd kuv yuav ntsuas qhov concentration tom qab ntxuav;ntawm no, Foregene xav kom nco ntsoov tias cDNA tsis pom zoo kom ua kom huv, vim tias qhov ntev ntawm cDNA tau txais los ntawm kev thim rov qab yog qhov sib txawv, thiab luv luv cDNA yuav ploj hauv kev ua kom huv.
(2) Yog li yuav ua li cas?Ua ntej qhov kev sim qPCR, qhov dilution gradient ntawm cDNA tuaj yeem txiav txim siab los ntawm kev sim ua ntej.Piv txwv li: siv cDNA Tshuag tov, 10-fold dilution, thiab 100-fold dilution raws li cov qauv rau qPCR thwmsim, thiab xaiv qhov dilution factor nrog tus nqi CT hauv thaj tsam ntawm 18-28.

Yuav ua li cas yuav tsum tau miRNAs reverse transcribed?
miRNA yog ib leeg-stranded me me molecule RNA nrog ib tug loj ntawm txog 22 nt uas tsis code rau protein.Vim hais tias ntawm nws luv luv, cov qauv qPCR yog ib qho nyuaj rau ncaj qha quantify nws, yog li nws yog feem ntau yuav tsum tau mus cuag miRNA;feem ntau siv cov kev hloov pauv rov qab rau miRNA suav nrog stem-loop method thiab tailing method.
Txoj kev qia-loop yog txhawm rau txuas lub miRNA los ntawm kev ntxiv qia-loop primers.Txoj kev tshawb nrhiav no muaj qhov rhiab heev thiab qhov tshwj xeeb, tab sis qhov kev kuaj pom tau qis dua.Ib daim ntawv rov qab tsuas tuaj yeem ntes tau ib qho miRNA thiab ib qho kev siv sab hauv;tus Tsov tus tw-ntxiv txoj kev yog tsim los ntawm ob Nws yog ua tiav los ntawm kev sib koom tes ntawm ob enzymes, uas yog PolyA polymerase thiab thim rov qab transcriptase.PolyA polymerase yog lub luag haujlwm rau ntxiv PolyA tails rau miRNA kom nws ntev, thiab thim rov qab transcriptase ua cov tshuaj tiv thaiv rov qab.Txoj kev no muaj kev tshawb pom siab dhau los thiab tuaj yeem tshawb xyuas ntau yam miRNAs thiab cov ntaub ntawv sab hauv hauv ib qho kev hloov pauv rov qab, tab sis qhov rhiab heev thiab qhov tshwj xeeb yog tsawg hauv txoj kev qia-voj.