Cov khoom pib: RNA
Quantitative reverse transcription PCR (RT-qPCR) yog ib txoj kev sim siv hauv PCR kev sim siv RNA ua cov khoom pib.Hauv txoj kev no, tag nrho RNA lossis tus xa xov RNA (mRNA) yog thawj zaug hloov mus rau hauv cov DNA sib ntxiv (cDNA) los ntawm thim rov qab transcriptase.Tom qab ntawd, cov tshuaj tiv thaiv qPCR tau ua tiav siv cDNA ua tus qauv.RT-qPCR tau siv ntau yam kev siv tshuaj lom neeg molecular, suav nrog kev tshuaj ntsuam gene qhia, RNA cuam tshuam validation, microarray validation, kuaj kab mob, kuaj caj ces, thiab kev tshawb fawb kab mob.
Ib-kauj ruam thiab ob-kauj ruam txoj kev rau RT-qPCR
RT-qPCR tuaj yeem ua tiav los ntawm ib kauj ruam lossis ob kauj ruam.Ib-kauj ruam RT-qPCR muab cov lus rov qab thiab PCR amplification, tso cai rau rov qab transcriptase thiab DNA polymerase ua kom tiav cov tshuaj tiv thaiv hauv tib lub raj nyob rau hauv tib yam tsis zoo.Ib-kauj ruam RT-qPCR tsuas yog yuav tsum tau siv cov txheej txheem tshwj xeeb primers.Nyob rau hauv ob-kauj ruam RT-qPCR, thim rov qab thiab PCR amplification yog ua nyob rau hauv ob lub raj, siv txawv optimized buffers, tshuaj tiv thaiv tej yam kev mob, thiab primer tsim tswv yim.
| Qhov zoo | Qhov tsis zoo | |
| Ib kauj ruam | Txoj kev no muaj tsawg dua qhov kev sim ua yuam kev vim tias ob qho tib si tshwm sim hauv ib lub raj
Tsawg dua cov kauj ruam pipetting txo qhov kev pheej hmoo ntawm kev kis kab mob
Haum rau high-throughput amplification / soj ntsuam, ceev thiab rov tsim dua | Cov tshuaj tiv thaiv ob-kauj ruam tsis tuaj yeem ua kom zoo sib cais
Txij li thaum cov kev mob tshwm sim raug cuam tshuam los ntawm kev sib txuas cov tshuaj tiv thaiv ob-kauj ruam, qhov rhiab heev tsis zoo li ntawm txoj kev ob-kauj ruam
Tus naj npawb ntawm cov hom phiaj kuaj pom los ntawm ib qho qauv me me |
| Ob kauj ruam | Muaj peev xwm tsim cov tsev qiv ntawv cDNA ruaj khov uas tuaj yeem khaws cia rau lub sijhawm ntev thiab siv ntau yam kev cuam tshuam
Lub hom phiaj cov noob thiab cov noob siv tau tuaj yeem nthuav dav los ntawm tib lub tsev qiv ntawv cDNA yam tsis tas yuav muaj ntau lub tsev qiv ntawv cDNA
Cov tshuaj tiv thaiv buffers thiab cov tshuaj tiv thaiv kab mob uas ua kom zoo tshaj plaws ntawm cov tshuaj tiv thaiv ib leeg
Saj zawg zog xaiv ntawm qhov ua rau tej yam kev mob | Kev siv ntau lub raj, thiab ntau cov txheej txheem pipetting ua rau muaj kev pheej hmoo ntawm DNA kis, thiab siv sijhawm.
Yuav tsum tau optimization ntau tshaj li ib-kauj ruam txoj kev |
Yam khoom:
RT-qPCR Easyᵀᴹ (Ib kauj ruam)-SYBR Green I
RT-qPCR Easyᵀᴹ (Ib kauj ruam)-Taqman
RT Easyᵀᴹ Kuv Master Premix Rau Thawj-Strand CDNA Synthesis
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Lub Sijhawm PCR Yooj Yim Yooj Yim Yooj Yim-Taqman
Xaiv tag nrho RNA thiab mRNA
Thaum tsim ib qho kev sim RT-qPCR, nws yog ib qho tseem ceeb uas yuav tau txiav txim siab seb puas yuav siv tag nrho RNA los yog purified mRNA raws li tus qauv rau kev hloov pauv rov qab.Txawm hais tias mRNA tuaj yeem muab qhov siab dua me ntsis, tag nrho RNA tseem nquag siv.Qhov laj thawj rau qhov no yog tias tag nrho RNA muaj qhov txiaj ntsig tseem ceeb dua li cov khoom pib dua li mRNA.Ua ntej, cov txheej txheem yuav tsum muaj tsawg dua cov kauj ruam ua kom huv, uas ua kom muaj txiaj ntsig zoo dua ntawm cov qauv thiab kev ua kom zoo dua qub ntawm cov txiaj ntsig los pib cov xov tooj ntawm tes.Qhov thib ob, nws zam qhov mRNA enrichment kauj ruam, uas tuaj yeem zam qhov muaj peev xwm ntawm skewed cov txiaj ntsig vim muaj qhov sib txawv ntawm cov mRNA sib txawv.Zuag qhia tag nrho, txij li nyob rau hauv feem ntau daim ntawv thov cov txheeb ze kom muaj nuj nqis ntawm lub hom phiaj noob tseem ceeb tshaj qhov tseeb rhiab heev ntawm kev kuaj pom, tag nrho RNA yog tsim nyog nyob rau hauv feem ntau.
Rov qab transcription primer
Hauv txoj kev ob-kauj ruam, peb txoj kev sib txawv tuaj yeem siv los ua cov tshuaj tiv thaiv cDNA: oligo (dT) primers, random primers, lossis sequence-specific primers.Feem ntau, oligo (dT) primers thiab random primers yog siv ua ke.Cov primers anneal rau tus qauv mRNA strand thiab muab rov qab transcriptase nrog rau qhov pib rau kev sib txuas.
| Primer xaiv | Cov qauv thiab kev ua haujlwm | Qhov zoo | Qhov tsis zoo |
| Oligo (dT) primer (los yog anchored oligo (dT) primer) | Txuas ntxiv mus rau thymine residues ntawm poly(A) tail ntawm mRNA;thauj tog rau nkoj oligo (dT) primer muaj G, C, lossis A ntawm 3′ kawg (lub chaw thauj tog rau nkoj) | Synthesis of full-length cDNA los ntawm poly(A)-tailed mRNA
Siv tau thaum muaj cov khoom pib tsawg dua
Anchoring site xyuas kom meej tias oligo (dT) primer khi rau 5′ poly (A) tail ntawm mRNA | Tsuas yog tsim rau amplifying noob nrog poly(A) tails
Tau txais cDNA txiav los ntawm qhov chaw priming * 2 hauv poly(A)
Biased los khi rau 3' kawg *
* Qhov no yuav txo tau yog tias siv cov oligo (dT) primers |
| random primer
| 6 mus rau 9 lub hauv paus ntev, uas tuaj yeem ua rau ntau qhov chaw thaum RNA transcription | Ntxiv rau txhua RNAs (tRNA, rRNA, thiab mRNA)
Haum rau cov ntawv sau nrog cov qauv tseem ceeb thib ob, lossis thaum muaj cov khoom pib tsawg dua
High cDNA Yield | cDNA yog thim rov qab los ntawm tag nrho cov RNA, uas feem ntau tsis xav tau thiab yuav dilute lub teeb liab ntawm lub hom phiaj mRNA.
tau truncated cDNA |
| ib ntus tshwj xeeb primers | Kev cai primers tsom rau mRNA ib ntus | ib lub tsev qiv ntawv cDNA
Txhim kho rhiab heev
Siv rov qab qPCR primers | Tsuas yog txwv rau cov synthesis ntawm ib lub hom phiaj gene |
Rov qab transcriptase
Reverse transcriptase yog ib qho enzyme uas siv RNA los tsim DNA.Qee qhov kev thim rov qab rov qab muaj cov haujlwm RNase thiab tuaj yeem txo qis RNA strands hauv RNA-DNA hybrid strands tom qab kev sau ntawv.Yog tias nws tsis muaj RNase enzymatic kev ua ub no, RNaseH tuaj yeem ntxiv rau qPCR ntau dua.Feem ntau siv cov enzymes suav nrog Moloney murine leukemia tus kab mob thim rov qab transcriptase thiab avian myeloblastoma tus kab mob thim rov qab transcriptase.Rau RT-qPCR, nws yog qhov zoo tshaj plaws los xaiv qhov rov qab transcriptase nrog cov thermostability siab dua, kom cDNA synthesis tuaj yeem ua tiav ntawm qhov kub siab dua, ua kom muaj txiaj ntsig zoo ntawm RNAs nrog cov qauv qib siab dua, thaum tuav lawv cov dej num tag nrho thoob plaws cov tshuaj tiv thaiv, ua rau cDNA ntau dua yields.
Yam khoom:
Foreasy M-MLV Reverse Transcriptase
RNase H kev ua haujlwm ntawm thim rov qab transcriptase
RNaseH muaj peev xwm degrade RNA strands los ntawm RNA-DNA duplexes, tso cai rau cov synthesis zoo ntawm ob-stranded DNA.Txawm li cas los xij, thaum siv mRNA ntev ua tus qauv, RNA yuav raug degraded ntxov ntxov, ua rau cDNA raug txiav.Yog li, nws feem ntau tau txais txiaj ntsig los txo qis RNaseH kev ua haujlwm thaum lub sijhawm cDNA cloning yog tias xav tau kev sib txuas ntawm cov ntawv sau ntev.Nyob rau hauv sib piv, thim rov qab transcriptases nrog RNase H kev ua ub no feem ntau muaj txiaj ntsig zoo rau cov ntawv thov qPCR vim tias lawv txhim kho cov melting ntawm RNA-DNA duplexes thaum thawj lub voj voog ntawm PCR.
Primer tsim
PCR primers siv rau qPCR kauj ruam hauv RT-qPCR yuav tsum tau tsim kom muaj kev sib txuas ntawm exon-exon junction, qhov twg amplification primer yuav muaj feem cuam tshuam qhov exon-intron ciam teb.Txij li thaum intron-muaj genomic DNA sequences tsis yog amplified, qhov kev tsim no txo qhov kev pheej hmoo ntawm qhov tsis tseeb qhov zoo los ntawm cov kab mob genomic DNA.
Yog tias primers tsis tuaj yeem tsim los cais exons lossis exon-exon ciam teb, nws yuav tsim nyog los kho RNA cov qauv nrog RNase-dawb DNase I lossis dsDNase kom tshem tawm cov kab mob DNA genomic.
RT-qPCR tswj
Kev tswj xyuas qhov tsis zoo rov qab (-RT tswj) yuav tsum muaj nyob rau hauv txhua qhov kev sim RT-qPCR txhawm rau txheeb xyuas DNA cov kab mob sib kis (xws li genomic DNA lossis PCR cov khoom los ntawm cov tshuaj tiv thaiv yav dhau los).Cov kev tswj no muaj tag nrho cov tshuaj tiv thaiv tshwj tsis yog rov qab transcriptase.Txij li qhov kev hloov pauv rov qab tsis tshwm sim nrog qhov kev tswj hwm no, yog tias pom muaj PCR amplification, kev kis kab mob los ntawm DNA feem ntau yuav.
Post lub sij hawm: Aug-02-2022
