Zoyambira: RNA
Quantitative reverse transcription PCR (RT-qPCR) ndi njira yoyesera yomwe imagwiritsidwa ntchito poyesa PCR pogwiritsa ntchito RNA monga poyambira.Munjira iyi, RNA yonse kapena messenger RNA (mRNA) imalembedwa koyamba kukhala DNA yowonjezera (cDNA) ndi reverse transcriptase.Pambuyo pake, qPCR reaction idachitika pogwiritsa ntchito cDNA ngati template.RT-qPCR yakhala ikugwiritsidwa ntchito m'machitidwe osiyanasiyana a biology ya mamolekyulu, kuphatikiza kusanthula kwa jini, kutsimikizira kusokoneza kwa RNA, kutsimikizika kwa microarray, kuzindikira kwa tizilombo toyambitsa matenda, kuyezetsa majini, ndi kafukufuku wa matenda.
Njira imodzi ndi njira ziwiri za RT-qPCR
RT-qPCR ikhoza kukwaniritsidwa ndi njira imodzi kapena ziwiri.Gawo limodzi la RT-qPCR limaphatikiza zolembera zobwerera kumbuyo ndi kukulitsa kwa PCR, kulola reverse transcriptase ndi DNA polymerase kuti amalize zomwe akuchita mu chubu yomweyo pansi pamikhalidwe yofananira.Gawo limodzi la RT-qPCR limangofunika kugwiritsa ntchito zoyambira zotsatizana.Mu masitepe awiri a RT-qPCR, kulembera kumbuyo ndi kukulitsa kwa PCR kumachitika m'machubu awiri, pogwiritsa ntchito ma buffers osiyanasiyana, momwe amachitira, ndi njira zoyambira.
| Ubwino | Kuipa | |
| Gawo Limodzi | Njirayi ili ndi zolakwika zochepa zoyesera chifukwa zonse zimachitika mu chubu chimodzi
Mapaipi ochepa amachepetsa chiopsezo cha kuipitsidwa
Oyenera kukulitsa / kuwunika kwapamwamba, mwachangu komanso kupangikanso | Zochita za magawo awiri sizingakwaniritsidwe padera
Popeza momwe machitidwe amasokonezedwa ndikuphatikiza njira ziwirizi, kukhudzikako sikuli bwino ngati njira yanjira ziwiri.
Chiwerengero cha zolinga zomwe zapezeka ndi chitsanzo chimodzi ndizochepa |
| Masitepe Awiri | Kutha kupanga malaibulale okhazikika a cDNA omwe amatha kusungidwa kwa nthawi yayitali ndikugwiritsidwa ntchito mosiyanasiyana.
Mitundu yomwe mukufuna komanso ma jini ofotokozera amatha kukulitsidwa kuchokera ku laibulale yomweyo ya cDNA popanda kufunikira kwa malaibulale angapo a cDNA.
Ma buffers ndi machitidwe omwe amathandizira kukhathamiritsa kwamayendedwe amodzi
Kusankha kosinthika kwazinthu zoyambitsa | Kugwiritsa ntchito machubu angapo, komanso njira zambiri zamapaipi kumawonjezera chiopsezo cha kuipitsidwa kwa DNA, ndi kuwononga nthawi.
Pamafunika kukhathamiritsa kwambiri kuposa njira imodzi yokha |
Zogwirizana nazo:
RT-qPCR Easyᵀᴹ (Chigawo Chimodzi) -SYBR Green I
RT-qPCR Easyᵀᴹ (Chigawo Chimodzi) -Taqman
RT Easyᵀᴹ I Master Premix Yoyamba-Strand CDNA Synthesis
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Kusankhidwa kwa RNA yonse ndi mRNA
Popanga kuyesa kwa RT-qPCR, ndikofunikira kusankha ngati mugwiritse ntchito RNA yonse kapena mRNA yoyeretsedwa ngati template yolembera mobwerera.Ngakhale mRNA ikhoza kupereka chidwi chokwera pang'ono, RNA yonse imagwiritsidwabe ntchito mobwerezabwereza.Chifukwa cha izi ndikuti RNA yonse ili ndi mwayi wofunikira ngati choyambira kuposa mRNA.Choyamba, ndondomekoyi imafuna masitepe ochepa oyeretsera, omwe amaonetsetsa kuti template ikuyenda bwino komanso kusinthika bwino kwa zotsatira kuti muyambe manambala a cell.Chachiwiri, imapewa njira yolemeretsa ya mRNA, yomwe ingapewe kuthekera kwa zotsatira zopotoka chifukwa cha kuchira kosiyanasiyana kwa ma mRNA osiyanasiyana.Ponseponse, popeza m'ntchito zambiri kuchuluka kwa jini yomwe mukufuna ndi yofunika kwambiri kuposa kukhudzidwa kwathunthu kwa kuzindikira, RNA yonse ndiyoyenera nthawi zambiri.
Reverse transcription primer
Munjira ziwirizi, njira zitatu zosiyana zitha kugwiritsidwa ntchito poyambitsa cDNA reaction: oligo(dT) zoyambira, zoyambira mwachisawawa, kapena zoyambira zotsatizana.Nthawi zambiri, zoyambira za oligo(dT) ndi zoyambira mwachisawawa zimagwiritsidwa ntchito kuphatikiza.Zoyambira izi zimalumikizana ndi template ya mRNA strand ndikupereka reverse transcriptase yokhala ndi poyambira poyambira.
| Kusankha koyambira | Kapangidwe ndi ntchito | Ubwino | Kuipa |
| Oligo(dT) primer (kapena anchored oligo(dT) primer) | Kuonjezera annealing kwa zotsalira thymine pa poly(A) mchira wa mRNA;anchor oligo(dT) primer ili ndi G, C, kapena A kumapeto kwa 3′ (malo a nangula) | Kaphatikizidwe ka cDNA yayitali kuchokera ku poly(A)-tailed mRNA
Imagwira ntchito ngati zoyambira zochepa zilipo
Malo oyikapo amaonetsetsa kuti choyambira cha oligo(dT) chimangirira ku 5′ poly(A) mchira wa mRNA. | Oyenera kokha kukulitsa majini okhala ndi michira ya poly(A).
Pezani cDNA yodulidwa kuchokera pamalo oyamba *2 mu poly(A)
Zokondera kumangirira mpaka 3 ′ malekezero *
*Kuthekera uku kumachepetsedwa ngati zoyambira za oligo(dT) zikugwiritsidwa ntchito |
| mwachisawawa choyambirira
| Maziko 6 mpaka 9 kutalika, omwe amatha kulumikizana ndi masamba angapo panthawi yolemba RNA | Anneal ku ma RNA onse (tRNA, rRNA, ndi mRNA)
Zoyenera zolembedwa zokhala ndi mawonekedwe achiwiri, kapena pomwe zoyambira zochepa zilipo
Kuchuluka kwa cDNA | cDNA imatembenuzidwa kuchokera ku RNA yonse, yomwe nthawi zambiri sichifunidwa ndipo imatha kuchepetsa chizindikiro cha mRNA chandamale.
kupeza cDNA yochepa |
| zoyambira zotsatizana | Zoyambira zamakasitomala zomwe zimayang'ana machitidwe ena a mRNA | laibulale ya cDNA yeniyeni
Limbikitsani chidwi
Kugwiritsa ntchito reverse qPCR zoyambira | Zimangotengera kaphatikizidwe ka jini imodzi ya chandamale |
Reverse transcriptase
Reverse transcriptase ndi puloteni yomwe imagwiritsa ntchito RNA kupanga DNA.Ma reverse transcriptases ena amakhala ndi zochita za RNase ndipo amatha kusokoneza zingwe za RNA mu RNA-DNA hybrid strand pambuyo polemba.Ngati ilibe enzymatic ya RNase, RNaseH ikhoza kuwonjezeredwa kuti igwire bwino ntchito ya qPCR.Ma enzyme omwe amagwiritsidwa ntchito kwambiri ndi Moloney murine leukemia virus reverse transcriptase ndi avian myeloblastoma virus reverse transcriptase.Kwa RT-qPCR, ndikwabwino kusankha reverse transcriptase yokhala ndi thermostability yapamwamba, kuti kaphatikizidwe ka cDNA kachitidwe kotentha kwambiri, kuwonetsetsa kuti kulembedwa bwino kwa ma RNA okhala ndi mawonekedwe apamwamba achiwiri, ndikusunga ntchito yawo yonse panthawi yonseyi, zomwe zimapangitsa kuti cDNA ikhale yochuluka.
Zogwirizana nazo:
Foreasy M-MLV Reverse Transcriptase
Ntchito ya RNase H ya reverse transcriptase
RNaseH imatha kusokoneza zingwe za RNA kuchokera ku RNA-DNA duplexes, kulola kuti kaphatikizidwe koyenera ka DNA yazingwe ziwiri.Komabe, mukamagwiritsa ntchito mRNA yayitali ngati template, RNA ikhoza kuchepetsedwa msanga, zomwe zimapangitsa kuti cDNA ikhale yocheperako.Chifukwa chake, nthawi zambiri zimakhala zopindulitsa kuchepetsa zochitika za RNaseH panthawi ya cDNA cloning ngati kaphatikizidwe kazolemba zazitali kakufunika.Mosiyana ndi izi, zolembera zosinthika zokhala ndi zochitika za RNase H nthawi zambiri zimakhala zopindulitsa pamapulogalamu a qPCR chifukwa zimawonjezera kusungunuka kwa ma RNA-DNA duplexes panthawi yoyamba ya PCR.
Kupanga koyambira
Zoyambira za PCR zomwe zimagwiritsidwa ntchito pa gawo la qPCR mu RT-qPCR ziyenera kupangidwa kuti zizikhala ndi mphambano ya exon-exon, pomwe choyambira chokulitsa chimatha kupitilira malire enieni a exon-intron.Popeza ma intron omwe ali ndi ma genomic DNA sakukwezedwa, kapangidwe kameneka kamachepetsa chiopsezo cha zolakwika zomwe zimachulukitsidwa kuchokera ku kuipitsa DNA ya genomic.
Ngati zoyambira sizingapangidwe kuti zilekanitse malire a ma exon kapena exon-exon, pangakhale koyenera kuchitira zitsanzo za RNA ndi RNase-free DNase I kapena dsDNase kuchotsa kuipitsidwa kwa genomic DNA.
Kuwongolera kwa RT-qPCR
Reverse transcript negative control (-RT control) iyenera kuphatikizidwa muzoyesa zonse za RT-qPCR kuti zizindikire kuipitsidwa kwa DNA (monga genomic DNA kapena zinthu za PCR kuchokera pazomwe zidachitika kale).Chiwongolerochi chili ndi zigawo zonse zochitira kupatula reverse transcriptase.Popeza kulembedwa kwa reverse sikuchitika ndi chiwongolero ichi, ngati kukulitsa kwa PCR kumawonedwa, kuipitsidwa kuchokera ku DNA ndikotheka.
Nthawi yotumiza: Aug-02-2022
