IB 、 Ua kom qhov rhiab heev ntawm cov tshuaj tiv thaiv kab mob:

1. Tshem tawm RNA zoo:

Ua tiav cDNA synthesis los ntawm cov khoom zoo RNA.Qhov zoo RNA yuav tsum tsawg kawg yog tag nrho-ntev thiab tsis pub rov qab transcriptase inhibitors xws li EDTA lossis SDS.Qhov zoo ntawm RNA txiav txim siab ntau npaum li cas cov ntaub ntawv uas koj tuaj yeem sau rau hauv cDNA.Ib hom RNA purification txoj kev yog ib txoj hauv kev siv guanidine isothiocyanate / acid phenol.Txhawm rau tiv thaiv kev kis kab mob los ntawm cov lej ntawm RNase, RNA cais tawm los ntawm cov qauv RNase-nplua nuj (xws li pancreas) yuav tsum tau khaws cia rau hauv formaldehyde kom khaws cov RNA zoo, tshwj xeeb tshaj yog rau kev khaws cia ntev.RNA rho tawm los ntawm nas lub siab yog qhov pib degraded tom qab muab cia rau hauv dej rau ib lub lis piam, thaum lub RNA rho tawm los ntawm nas spleen nyob ruaj khov tom qab muab cia rau hauv dej rau 3 xyoos.Tsis tas li ntawd, cov ntawv sau tseg ntev dua 4 kb yog qhov nkag siab ntau dua rau kev degradation los ntawm kab RNases dua li cov ntawv sau me me.Txhawm rau kom muaj kev ruaj ntseg ntawm cov qauv khaws cia RNA, RNA tuaj yeem yaj hauv deionized formamide thiab khaws cia ntawm -70 ° C.Formamide siv los khaws RNA yuav tsum tsis pub RNA-degrading khib nyiab.RNA los ntawm pancreas tuaj yeem khaws cia hauv formamide tsawg kawg ib xyoos.Thaum npaj siv RNA, koj tuaj yeem siv cov txheej txheem hauv qab no txhawm rau txhawm rau RNA: ntxiv NaCl rau 0.2M thiab 4 npaug ntawm cov ntim ntawm ethanol, tso rau hauv chav sov li 3-5 feeb, thiab centrifuge ntawm 10,000 × g rau 5 feeb.

2. Siv RNaseH-inactive (RNaseH-) thim rov qab transcriptase:

RNase inhibitors feem ntau ntxiv rau cov kev hloov pauv rov qab los ua kom qhov ntev thiab tawm los ntawm cDNA synthesis.RNase inhibitors yuav tsum tau ntxiv thaum lub sij hawm thawj-strand synthesis cov tshuaj tiv thaiv nyob rau hauv lub xub ntiag ntawm ib tug tsis thiab ib tug txo tus neeg sawv cev (xws li DTT), vim hais tias cov txheej txheem ua ntej cDNA synthesis denatures tus inhibitor, yog li tso tseg RNase uas yuav degrade RNA.Protein RNase inhibitors tsuas yog tiv thaiv kev degradation ntawm RNA los ntawm RNase A, B, C, thiab tsis tiv thaiv RNase ntawm daim tawv nqaij, yog li ceev faj tsis txhob qhia RNase los ntawm koj cov ntiv tes txawm tias siv cov inhibitors.

Rov qab transcriptase catalyzes kev hloov pauv ntawm RNA rau cDNA.Ob leeg M-MLV thiab AMV muaj cov haujlwm endogenous RNaseH ntxiv rau lawv tus kheej cov haujlwm polymerase.RNaseH kev ua haujlwm thiab cov haujlwm polymerase sib tw ua ke rau kev sib xyaw ua ke ntawm RNA template thiab DNA primer lossis cDNA extension strand, thiab degrade RNA strand hauv RNA: DNA complex.RNA template degraded los ntawm RNaseH kev ua ub no tsis tuaj yeem ua ib qho zoo substrate rau cDNA synthesis, uas txo cov yield thiab ntev ntawm cDNA synthesis.Yog li, nws yuav muaj txiaj ntsig zoo los tshem tawm lossis txo qis RNaseH kev ua haujlwm ntawm thim rov qab transcriptase..

SuperScript Ⅱ thim rov qab transcriptase, RNaseH- MMLV thim rov qab transcriptase thiab thermoScript thim rov qab transcriptase, RNaseH- AMV, tuaj yeem tau txais ntau npaum li cas thiab ntau qhov ntev cDNA tshaj MMLV thiab AMV.RT-PCR rhiab heev yuav raug cuam tshuam los ntawm tus nqi ntawm cDNA synthesis.ThermoScript yog rhiab heev dua AMV.Qhov loj ntawm RT-PCR cov khoom raug txwv los ntawm lub peev xwm ntawm kev rov qab transcriptase los ua ke cDNA, tshwj xeeb tshaj yog thaum cloning loj cDNAs.Piv nrog rau MMLV, SuperScripⅡ tau nce cov txiaj ntsig ntawm cov khoom RT-PCR ntev.RNaseH-reverse transcriptase kuj tseem muaj qhov kub thiab txias, yog li cov tshuaj tiv thaiv tuaj yeem ua rau kub siab tshaj li qhov qub 37-42 ° C.Nyob rau hauv cov kev qhia pom zoo, siv oligo (dT) primer thiab 10 μCi ntawm [α-P]dCTP.Tag nrho cov txiaj ntsig ntawm thawj strand tau suav nrog siv TCA txoj kev los nag.Tag nrho-ntev cDNA raug tshuaj xyuas los ntawm kev siv qhov loj-sorted bands excised thiab suav rau ntawm alkaline agarose gel.

3. Tsa qhov kub thiab txias rau kev hloov pauv rov qab:

Qhov kub incubation ntau dua pab qhib RNA cov qauv theem nrab, ua rau cov txiaj ntsig ntawm cov tshuaj tiv thaiv.Rau feem ntau RNA templates, incubating RNA thiab primers ntawm 65 ° C yam tsis muaj buffer los yog ntsev, ua raws li los ntawm ceev ceev txias ntawm cov dej khov yuav tshem tawm feem ntau cov qauv thiab cia primers khi.Txawm li cas los xij, qee cov qauv tseem muaj cov txheej txheem thib ob, txawm tias tom qab cua sov denaturation.Kev nthuav dav ntawm cov qauv nyuaj no tuaj yeem ua tau siv ThermoScript Reverse Transcriptase thiab tso cov lus rov qab rov qab los ntawm qhov kub siab dua txhawm rau txhim kho kev nthuav dav.Kev kub hnyiab ntau dua tuaj yeem ua rau muaj qhov tshwj xeeb, tshwj xeeb tshaj yog thaum cov gene-specific primers (GSP) siv rau cDNA synthesis (saib Tshooj 3).Yog tias siv GSP, xyuas kom meej tias Tm ntawm cov primers yog tib yam li qhov xav tau qhov kub thiab txias.Tsis txhob siv oligo (dT) thiab random primers saum 60 ° C.Random primers yuav tsum tau incubation ntawm 25 ° C rau 10 feeb ua ntej nce mus rau 60 ° C.Ntxiv nrog rau kev siv qhov kub thiab txias dua, qhov tshwj xeeb kuj tuaj yeem txhim kho los ntawm kev xa ncaj qha RNA / primer mix los ntawm 65 ° C denaturation kub mus rau qhov rov qab transcription incubation kub thiab ntxiv pre-warmed 2 × cov tshuaj tiv thaiv sib tov (cDNA kub-pib synthesis).Txoj hauv kev no pab tiv thaiv kev sib koom ua ke ntawm intermolecular puag uas tshwm sim ntawm qhov kub thiab txias.Kev hloov pauv ntau yam uas xav tau rau RT-PCR tuaj yeem ua kom yooj yim los ntawm kev siv lub thermal cycler.

Tth thermostable polymerase ua raws li DNA polymerase nyob rau hauv lub xub ntiag ntawm Mg2 + thiab raws li ib tug RNA polymerase nyob rau hauv lub xub ntiag ntawm Mn2 +.Nws tuaj yeem khaws cia sov ntawm qhov kub siab tshaj ntawm 65 ° C.Txawm li cas los xij, lub xub ntiag ntawm Mn2+ thaum PCR txo kev ncaj ncees, uas ua rau Tth polymerase tsis haum rau kev ua kom pom tseeb, xws li cloning ntawm cDNA.Tsis tas li ntawd, Tth muaj qhov rov qab rov qab ua haujlwm tsis zoo, uas txo qhov kev xav tau, thiab, txij li rov qab hloov pauv thiab PCR tuaj yeem ua tau nrog ib qho enzyme, kev tswj cov tshuaj tiv thaiv tsis tuaj yeem siv los sib piv cDNA amplification khoom nrog cov kab mob genomic DNA.Cov khoom lag luam amplification tau sib cais.

4. Additives uas txhawb kev thim rov qab:

Cov tshuaj ntxiv nrog rau glycerol thiab DMSO tau ntxiv rau thawj-strand synthesis cov tshuaj tiv thaiv, uas tuaj yeem txo qhov kev ruaj ntseg ntawm nucleic acid ob-strand thiab untie tus qauv thib ob ntawm RNA.Txog li 20% glycerol lossis 10% DMSO tuaj yeem ntxiv yam tsis muaj kev cuam tshuam rau SuperScript II lossis MMLV kev ua haujlwm.AMV kuj tuaj yeem zam tau txog li 20% glycerol yam tsis muaj kev poob haujlwm.Txhawm rau ua kom qhov siab tshaj plaws ntawm RT-PCR hauv SuperScriptⅡ rov qab cov tshuaj tiv thaiv, 10% glycerol tuaj yeem muab ntxiv thiab ua rau ntawm 45 ° C.Yog tias 1/10 ntawm cov khoom siv rov qab hloov pauv cov tshuaj tiv thaiv ntxiv rau PCR, tom qab ntawd cov ntsiab lus ntawm glycerol hauv cov tshuaj tiv thaiv amplification yog 0.4%, uas tsis txaus rau inhibit PCR.

5. RNaseH kho:

Kev kho ntawm cDNA synthesis cov tshuaj tiv thaiv nrog RNaseH ua ntej PCR tuaj yeem ua rau muaj kev nkag siab zoo.Rau qee cov qauv, nws tau xav tias RNA hauv cDNA synthesis cov tshuaj tiv thaiv tiv thaiv kev khi ntawm cov khoom lag luam amplification, qhov twg RNaseH kev kho mob tuaj yeem ua rau muaj kev nkag siab zoo.Feem ntau, kev kho RNaseH yog qhov tsim nyog thaum nthuav dav ntev ntev cDNA lub hom phiaj cov qauv, xws li cov ntawv theej qis tuberous Scherosis II.Rau cov qauv nyuaj no, kev kho RNaseH txhim kho lub teeb liab tsim los ntawm SuperScript II lossis AMV-synthesized cDNA.Rau feem ntau RT-PCR cov tshuaj tiv thaiv, kev kho RNaseH yog xaiv tau, vim hais tias PCR denaturation kauj ruam ntawm 95 ° C feem ntau hydrolyzes RNA hauv RNA: DNA complex.

6. Kev Txhim Kho ntawm Me Me RNA Detection Method:

RT-PCR yog qhov nyuaj tshwj xeeb thaum tsuas yog me me ntawm RNA muaj.Glycogen ntxiv raws li tus neeg nqa khoom thaum lub sijhawm RNA cais pab ua kom cov txiaj ntsig ntawm cov qauv me me.RNase-dawb glycogen tuaj yeem muab ntxiv rau tib lub sijhawm thaum ntxiv Trizol.Glycogen yog dej soluble thiab tuaj yeem khaws cia rau hauv theem dej nrog RNA los pab nag lossis daus.Rau cov qauv tsawg dua 50 mg ntawm cov ntaub so ntswg los yog 106 kab lis kev cai, cov lus pom zoo ntawm RNase-dawb glycogen yog 250 μg / ml.

Ntxiv acetylated BSA rau qhov kev hloov pauv rov qab siv SuperScript II tuaj yeem ua rau muaj kev nkag siab ntau ntxiv, thiab rau qhov me me ntawm RNA, txo tus nqi ntawm SuperScript II thiab ntxiv 40 units ntawm RNaseOut nuclease inhibitor tuaj yeem nce qib ntawm kev tshawb pom.Yog tias siv glycogen hauv RNA kev cais tawm, nws tseem pom zoo kom ntxiv BSA lossis RNase inhibitor thaum siv SuperScript II rau cov tshuaj tiv thaiv rov qab.

、 Ua kom RT-PCR tshwj xeeb

1. CND Asynthesis:

Thawj-strand cDNA synthesis tuaj yeem pib siv peb txoj kev sib txawv, qhov txheeb ze qhov tshwj xeeb uas cuam tshuam rau tus nqi thiab hom cDNA synthesized.

Txoj kev random primer yog qhov tsawg tshaj plaws ntawm peb txoj kev.Primers anneal ntawm ntau qhov chaw thoob plaws hauv cov ntawv sau tseg, tsim kom luv, ib nrab-ntev cDNAs.Cov qauv no nquag siv kom tau txais 5′ qhov kawg ua ntu zus thiab tau txais cDNA los ntawm RNA cov qauv nrog thaj tsam ntawm cov qauv thib ob lossis nrog qhov chaw txiav tawm uas tsis tuaj yeem rov ua dua los ntawm kev rov qab transcriptase.Txhawm rau kom tau txais qhov ntev tshaj plaws cDNA, qhov piv ntawm primers rau RNA hauv txhua tus qauv RNA yuav tsum tau txiav txim siab empirically.Qhov pib concentration ntawm random primers ranges ntawm 50 mus rau 250 ng ib 20 μl cov tshuaj tiv thaiv.Txij li thaum cDNA synthesized los ntawm tag nrho RNA siv random primers feem ntau ribosomal RNA, poly(A) + RNA feem ntau yog xaiv raws li tus qauv.

Oligo (dT) primers yog tshwj xeeb tshaj yog random primers.Nws hybridizes mus rau poly(A) tus Tsov tus tw pom ntawm 3' kawg ntawm feem ntau eukaryotic mRNAs.Vim hais tias poly(A) + RNA yog kwv yees li 1% mus rau 2% ntawm tag nrho RNA, tus nqi thiab complexity ntawm cDNA yog tsawg dua nrog random primers.Vim tias nws qhov tshwj xeeb siab, oligo (dT) feem ntau tsis xav tau kev ua kom zoo ntawm qhov piv ntawm RNA rau primers thiab poly (A) + xaiv.Nws raug pom zoo kom siv 0.5μg oligo (dT) ib 20μl cov tshuaj tiv thaiv kab mob.oligo (dT) 12-18 yog haum rau feem ntau RT-PCR.ThermoScript RT-PCR System muaj oligo(dT)20 vim tias nws cov thermal stability zoo dua rau qhov kub hnyiab ntau dua.

Gene specific primers (GSP) yog cov primers tshwj xeeb tshaj plaws rau cov kauj ruam rov qab.GSP yog ib qho antisense oligonucleotide uas muaj peev xwm tshwj xeeb hybridize rau RNA lub hom phiaj sib lawv liag, tsis zoo li random primers los yog oligo (dT), uas anneal rau tag nrho cov RNAs.Tib txoj cai siv los tsim PCR primers siv rau kev tsim ntawm GSP hauv kev hloov pauv rov qab.GSP tuaj yeem ua tib yam raws li qhov kev nthuav dav ntxiv uas txuas mus rau 3'-feem ntau kawg ntawm mRNA, los yog GSP tuaj yeem tsim los rau anneal downstream ntawm qhov rov qab amplification primer.Rau qee qhov kev nthuav dav, ntau tshaj ib qho antisense primer yuav tsum tau tsim kom muaj kev vam meej RT-PCR vim tias cov qauv theem nrab ntawm lub hom phiaj RNA tuaj yeem tiv thaiv primer binding.Nws raug pom zoo kom siv 1 pmol antisense GSP hauv 20 μl thawj-strand synthesis cov tshuaj tiv thaiv.

2. Tsa lub incubation kub rau reverse transcription:

Txhawm rau kom tau txais txiaj ntsig zoo ntawm tag nrho cov txiaj ntsig ntawm GSP qhov tshwj xeeb, qhov rov qab transcriptase yuav tsum tau siv ntau dua thermostability.Thermostable thim rov qab transcriptases tuaj yeem tsim nyob rau ntawm qhov kub siab dua kom ua rau cov tshuaj tiv thaiv stringency.Piv txwv li, yog tias GSP anneals ntawm 55 ° C, qhov tshwj xeeb ntawm GSP yuav tsis siv tag nrho yog tias AMV lossis M-MLV yog siv rau kev thim rov qab ntawm qhov qis qis ntawm 37 ° C.Txawm li cas los xij, SuperScript II thiab ThermoScript tuaj yeem tshwm sim ntawm 50 ° C lossis siab dua, uas yuav tshem tawm cov khoom tsis tshwj xeeb uas tsim los ntawm qhov kub thiab txias.Rau qhov tshwj xeeb tshaj plaws, RNA / primer mix tuaj yeem xa ncaj qha los ntawm 65 ° C denaturation kub mus rau qhov rov qab transcription incubation kub thiab ntxiv rau pre-warmed 2 × cov tshuaj tiv thaiv mix (cDNA synthesis kub pib).Qhov no yuav pab tiv thaiv kev sib tshuam hauv nruab nrab ntawm qhov kub thiab txias.Ntau qhov kev hloov pauv kub uas xav tau rau RT-PCR tuaj yeem ua kom yooj yim los ntawm kev siv lub thermal cycler.

3. Txo cov kab mob DNA genomic:

Ib qho teeb meem uas tau ntsib nrog RT-PCR yog kev kis kab mob ntawm cov DNA genomic hauv RNA.Siv txoj kev sib cais RNA zoo, xws li Trizol Reagent, yuav txo cov genomic DNA uas kis tau RNA npaj.Txhawm rau zam cov khoom lag luam los ntawm genomic DNA, RNA tuaj yeem kho nrog kev nthuav dav-qib DNase I kom tshem tawm cov kab mob DNA ua ntej rov thim rov qab.Kev zom zaub mov DNase I tau raug txiav tawm los ntawm incubating cov qauv hauv 2.0 mM EDTA rau 10 feeb ntawm 65 ° C.EDTA tuaj yeem chelate magnesium ions, tiv thaiv magnesium ion-dependent RNA hydrolysis ntawm qhov kub thiab txias.

Txhawm rau cais cov cDNA amplified los ntawm cov kab mob genomic DNA amplification cov khoom, primers tuaj yeem tsim tau tias txhua qhov anneal cais exons.PCR cov khoom tau muab los ntawm cDNA yuav luv dua li cov khoom tau los ntawm cov kab mob DNA uas muaj kab mob.Tsis tas li ntawd, kev tswj xyuas qhov kev sim uas tsis muaj kev rov qab rov qab tau ua tiav ntawm txhua tus qauv RNA los txiav txim seb puas muaj cov khoom seem tau muab los ntawm genomic DNA lossis cDNA.Cov khoom PCR tau txais yam tsis muaj kev hloov pauv rov qab yog muab los ntawm genome.