Ⅰ. Ua kom qhov rhiab heev ntawm cov tshuaj tiv thaiv kab mob:

1. Sib cais zoo RNA:

Ua tiav cDNA synthesis los ntawm cov khoom zoo RNA.High-quality RNA yuav tsum xyuas kom tsawg kawg nkaus ntev dua thiab tsis muaj cov inhibitors uas tsis muaj cov enzymes kaw, xws li EDTA lossis SDS.Qhov zoo ntawm RNA txiav txim siab tus nqi siab tshaj plaws ntawm cov ntaub ntawv sib txuas uas koj tuaj yeem xa mus rau cDNA.Txoj kev ua kom huv RNA dav dav yog ib kauj ruam ntawm kev siv isoocyanate/acidophenol.Txhawm rau tiv thaiv kev ua qias tuaj ntawm RNase, RNA sib cais los ntawm cov qauv nplua nuj hauv RNase (xws li pancreas) yuav tsum tau khaws cov formaldehyde kom txuag tau cov khoom zoo RNA, uas yog ntau dua rau kev khaws cia ntev.RNA rho tawm los ntawm nas lub siab yog qhov pib degraded tom qab ib lub lim tiam ntawm kev cia hauv dej, thaum lub RNA rho tawm los ntawm nas spleen tseem nyob ruaj khov tom qab peb xyoos cia hauv dej.Tsis tas li ntawd, cov ntaub ntawv sau tseg loj dua 4kb yog qhov nkag siab zoo rau kev taug qab RNase degradation dua li cov ntawv sau me me.Txhawm rau kom muaj kev ruaj ntseg ntawm cov qauv cia RNA, RNA tuaj yeem yaj hauv cov tshuaj methalamine ntawm ion, thiab khaws cia -70 ° C.Thylide siv los cawm RNA yuav tsum tsis muaj lwm yam khoom uas degrades RNA.RNA, uas yog muab los ntawm cov txiav txiav, tuaj yeem khaws cia hauv cov tshuaj methalamine tsawg kawg ib xyoos.Thaum koj npaj txhij 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 qhov ntim ntawm ethanol, tso rau hauv chav sov li 3-5 feeb, thiab 10,000 × g centrifugal rau 5 feeb.

2. Siv cov ntawv thim rov qab yam tsis muaj RNaseH kev ua (RNaseH-):

RNase inhibitors feem ntau ntxiv rau cov kev hloov pauv rov qab los ua kom qhov ntev thiab tawm los ntawm cDNA synthesis.RNase inhibitor yog ntxiv rau hauv thawj cov saw synthesis cov tshuaj tiv thaiv nyob rau hauv lub xub ntiag ntawm buffers thiab txo cov neeg ua hauj lwm xws li DTT vim hais tias cov pre-cDNA synthesis txheej txheem denatures tus inhibitor, yog li tso tseg RNases uas degrade RNA.Protein RNase inhibitor tsuas yog tiv thaiv kev degradation ntawm RNA los ntawm RNase A, B, C, thiab tsis tiv thaiv RNases ntawm daim tawv nqaij, yog li kev saib xyuas yuav tsum tsis txhob qhia RNases los ntawm tus ntiv tes txawm tias siv cov inhibitors no.

Rov qab transcriptase catalyzes hloov 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 si sib tw nrog polymerase kev ua si rau heterozygous strands tsim ntawm RNA templates thiab DNA primers los yog cDNA extension strands, thiab degrades RNA: RNA strands nyob rau hauv DNA complexes.RNA templates degraded los ntawm RNaseH kev ua ub no tsis tuaj yeem siv los ua cov substrates zoo rau cDNA synthesis, txo cov yield thiab ntev ntawm cDNA synthesis.Yog li tshem tawm lossis txo qis RNaseH cov haujlwm ntawm kev thim rov qab transcriptase yuav muaj txiaj ntsig zoo.

SuperScriptⅡ thim rov qab transcriptase, MMLV thim rov qab transcriptase ntawm RNaseH- thiab thermoScript thim rov qab transcriptase, AMV ntawm RNaseH- tau txais txiaj ntsig ntau dua cDNA ntau dua li MMLV thiab AMV.RT-PCR rhiab heev yog cuam tshuam los ntawm tus nqi ntawm cDNA synthesized.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-'s thim rov qab transcriptase kuj nce thermal stability, yog li cov tshuaj tiv thaiv tuaj yeem nqa tawm ntawm qhov kub siab tshaj li qub ntawm 37-42 ℃.Raws li cov txheej txheem kev sib txuas lus pom zoo, oligo (dT) primers thiab 10μCi [alpha-p]dCTP tau siv.Tag nrho cov khoom lag luam ntawm thawj cov saw hlau tau suav nrog kev siv TCA nag lossis daus.Tag nrho-ntev cDNA tau soj ntsuam siv qhov loj-tsim strip tshem tawm thiab suav hauv alkaline agarose gel.

3. Ua kom cov cua sov preservation kub ntawm rov qab transcription:

Kev tuav kub siab dua yuav pab qhib cov qauv thib ob ntawm RNA thiab nce cov txiaj ntsig ntawm cov tshuaj tiv thaiv.Rau feem ntau RNA templates, tuav lub RNA thiab primer ntawm 65 ° C yam tsis muaj buffer los yog ntsev thiab ces txias lawv sai sai rau cov dej khov tshem tawm feem ntau cov qauv thiab tso cai rau cov primers khi.Txawm li cas los xij, qee cov qauv tseem muaj cov qauv thib ob, txawm tias tom qab thermal denaturation.Kev nthuav dav ntawm cov qauv nyuaj no tuaj yeem ua tau siv ThermoScript thim rov qab transcriptase thiab los ntawm kev tso qhov rov qab transcriptase cov tshuaj tiv thaiv ntawm qhov kub siab dua txhawm rau txhim kho amplification.Kev tuav kub siab dua tuaj yeem ua rau muaj qhov tshwj xeeb, tshwj xeeb tshaj yog thaum cDNA synthesis siv cov noob tshwj xeeb primers (GSPS) (saib Tshooj 3).Yog tias siv GSP, xyuas kom tseeb tias tus nqi Tm ntawm primer yog tib yam li qhov xav tau tuav.Tsis txhob siv oligo (dT) thiab random primers saum 60 ℃.Random primers yuav tsum tau tuav ntawm 25 ℃ rau 10 feeb ua ntej nce mus rau 60 ℃.Ntxiv nrog rau kev siv qhov ntsuas kub rov qab siab dua, qhov tshwj xeeb tuaj yeem txhim kho los ntawm kev xa ncaj qha RNA / primer sib tov los ntawm 65 ℃ denaturing kub rau qhov rov qab transcription tuav kub thiab ntxiv ib qho preheated 2 × cov tshuaj tiv thaiv sib tov (cDNA thermal 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.Siv cov cuab yeej PCR ua kom yooj yim rau ntau qhov ntsuas kub uas xav tau rau RT-PCR.

Tth tshav kub stabilized polymerase ua raws li DNA polymerase nyob rau hauv lub xub ntiag ntawm Mg2 + thiab RNA polymerase nyob rau hauv lub xub ntiag ntawm Mn2 +.Nws tuaj yeem tuav tau kub txog li 65 ℃.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 cDNA cloning.Tsis tas li ntawd, Tth tsis tshua muaj txiaj ntsig ntawm kev hloov pauv rov qab, uas txo qis kev nkag siab, thiab txij li ib qho enzyme tuaj yeem ua rov qab hloov pauv thiab PCR, kev tswj cov tshuaj tiv thaiv tsis tuaj yeem siv los txheeb xyuas cov khoom lag luam ntawm cDNA los ntawm cov kab mob genomic DNA.

4. Additive uas txhawb kev thim rov qab:

Qhov sib ntxiv ntawm cov tshuaj ntxiv, suav nrog glycerin thiab DMSO, rau thawj cov saw hlau synthesis cov tshuaj tiv thaiv tuaj yeem txo qhov kev ruaj ntseg ntawm nucleic acid ob strand thiab unwind RNA theem nrab qauv.Txog li 20% glycerin lossis 10% DMSO tuaj yeem ntxiv yam tsis muaj kev cuam tshuam rau kev ua haujlwm ntawm SuperScriptⅡ lossis MMLV.AMV tseem tuaj yeem zam tau txog li 20% glycerol yam tsis muaj kev txo qis kev ua haujlwm.Txhawm rau ua kom qhov siab tshaj plaws ntawm RT-PCR hauv SuperScriptⅡ thim rov qab cov tshuaj tiv thaiv, 10% glycerol tuaj yeem ntxiv thiab insulated ntawm 45 ℃.Yog hais tias 1/10 ntawm cov khoom retrotranscription-reaction yog ntxiv rau PCR, qhov concentration ntawm glycerol nyob rau hauv lub amplification cov tshuaj tiv thaiv yog 0.4%, uas yog tsis txaus rau inhibit PCR.

5. RNaseH ua:

rhiab heev tuaj yeem txhim kho los ntawm kev kho cDNA synthesis cov tshuaj tiv thaiv nrog RNaseH ua ntej PCR.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 amplified, nyob rau hauv cov ntaub ntawv no kev kho mob RNaseH tuaj yeem ua rau rhiab heev.Feem ntau, RNaseH kev kho mob yuav tsum tau ua kom muaj kev nthuav dav ntawm qhov ntev-ntev cDNA lub hom phiaj template, xws li tuberous scherosisⅡ nrog cov ntawv tsawg.Rau cov qauv nyuaj no, RNaseH txhim kho cov teeb liab tsim los ntawm cDNA synthesized los ntawm SuperScriptⅡ lossis AMV.Rau feem ntau RT-PCR cov tshuaj tiv thaiv, RNaseH kev kho yog xaiv tau vim tias 95 ℃ insulated PCR denaturation kauj ruam feem ntau hydrolyzes RNA los ntawm RNA: DNA complex.

6. Txhim kho txoj hauv kev txhawm rau txheeb xyuas qhov me me ntawm RNA:

RT-PCR yog qhov nyuaj tshwj xeeb thaum tsuas yog me me ntawm RNA muaj.Qhov sib ntxiv ntawm glycogen raws li tus neeg nqa khoom thaum lub sijhawm RNA sib cais pab ua kom cov qoob loo ntawm cov qauv me me.Ib qho RNase-dawb glycogen tuaj yeem ntxiv rau tib lub sijhawm li Trizol.Glycogen yog dej-soluble thiab tuaj yeem nyob twj ywm hauv cov dej theem nrog RNA los pab rau yav tom ntej nag lossis daus.Qhov pom zoo kom pom zoo ntawm RNase-dawb glycogen yog 250μg / ml rau cov qauv tsawg dua 50mg ntawm cov ntaub so ntswg lossis 106 kab lis kev cai.

Qhov sib ntxiv ntawm acetylated BSA los thim rov qab cov lus teb rov qab siv SuperScriptⅡ tuaj yeem ua rau muaj kev nkag siab ntau ntxiv, thiab rau qhov me me ntawm RNA, txo tus nqi ntawm SuperScriptⅡ thiab ntxiv 40 units ntawm RnaseOut nuclease inhibitor tuaj yeem txhim kho qib kev tshawb pom.Yog tias glycogen tau siv hauv RNA sib cais, qhov sib ntxiv ntawm BSA lossis RNase inhibitors kom thim rov qab cov lus pom zoo siv SuperScriptⅡ tseem pom zoo.

Ⅱ. Nce qhov tshwj xeeb ntawm RT-PCR

1. CNDA synthesis:

Peb txoj kev sib txawv tuaj yeem siv los pib ua thawj strand cDNA synthesis, thiab qhov txheeb ze ntawm txhua txoj kev cuam tshuam rau tus nqi thiab hom cDNA synthesized.

Random primer txoj kev yog qhov tsawg tshaj plaws ntawm peb txoj kev.Primers raug muab tshem tawm ntawm ntau qhov chaw thoob plaws hauv cov ntawv sau los tsim cov luv luv, ib nrab ntev cDNA.Txoj kev no feem ntau yog siv kom tau txais 5 'terminal sequences thiab cDNA los ntawm RNA templates nrog rau theem nrab structural cheeb tsam los yog nrog terminating qhov chaw uas reverse transcriptase replicate.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.Thawj qhov concentration ntawm random primers yog li ntawm 50 txog 250ng ib 20μl cov tshuaj tiv thaiv kab mob.Vim hais tias cov cDNA synthesized los ntawm tag nrho cov RNA siv random primers feem ntau yog ribosomal RNA, poly(A) + RNA feem ntau xaiv raws li tus qauv.

Oligo (dT) pib yog qhov tshwj xeeb tshaj li cov primers random.Nws hybridizes nrog poly (A) tus Tsov tus tw pom nyob rau ntawm 3′ kawg ntawm mRNA nyob rau hauv feem ntau eukaryotic hlwb.Vim tias poly(A) + RNA yog kwv yees li 1% mus rau 2% ntawm tag nrho RNA, tus nqi thiab qhov nyuaj ntawm cDNA tsawg dua yog siv cov primers random.Vim tias nws qhov tshwj xeeb siab, oligo (dT) feem ntau tsis xav tau kev ua kom zoo rau RNA rau primer piv 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 muab oligo (dT) 20 vim nws cov thermal stability zoo thiab tsim nyog rau kev tuav ntau dua.

Gene-specific primers (GSP) yog qhov zoo tshaj plaws tshwj xeeb primers rau cov kauj ruam rov qab.GSP yog ib qho antisense oligonucleoside uas tuaj yeem sib xyaw nrog RNA qhov chaw ua ntu zus, es tsis yog txhua tus Rnas zoo li random primers lossis oligo (dT).Cov kev cai siv los tsim PCR primers kuj tseem siv tau rau kev tsim cov tshuaj tiv thaiv rov qab GSP.GSP tuaj yeem ua tau zoo ib yam li cov amplification primer annealed ntawm qhov kawg ntawm mRNA3', los yog GSP tuaj yeem tsim los ua kom qis qis qis nrog qhov rov qab amplification primer.Rau qee cov khoom lag luam, nws yog ib qho tsim nyog los tsim ntau tshaj ib qho antisense primer rau kev ua tiav RT-PCR vim tias cov qauv theem nrab ntawm lub hom phiaj RNA tuaj yeem tiv thaiv cov primer los ntawm kev khi.Nws raug nquahu kom siv 1pmol antisense GSP hauv thawj cov saw hlau synthesis cov tshuaj tiv thaiv ntawm 20μl.

2. Ua kom cov cua sov preservation ntawm rov qab transcription:

Txhawm rau kom tau txais txiaj ntsig zoo ntawm GSP qhov tshwj xeeb, rov qab transcriptase nrog siab thermal stability yuav tsum tau siv.Thaum tshav kub kub-ruaj khov rov qab transcriptase tuaj yeem raug rwb thaiv tsev ntawm qhov kub thiab txias kom nce cov tshuaj tiv thaiv nruj.Piv txwv li, yog tias GSP yog annealed ntawm 55 ° C, ces qhov tshwj xeeb ntawm GSP yog tsis siv tag nrho yog hais tias rov qab transcription yog ua ntawm 37 ° C nrog tsawg nruj siv AMV los yog M-MLV.Txawm li cas los xij, SuperScripⅡ thiab ThermoScript tuaj yeem hnov ​​​​qab ntawm 50 ℃ lossis siab dua, uas tshem tawm cov khoom tsis tshwj xeeb uas ua rau qis dua.Rau qhov tshwj xeeb tshaj plaws, RNA / primer sib tov tuaj yeem xa ncaj qha los ntawm 65 ℃ denaturation kub mus rau qhov rov qab hloov pauv tuav kub nrog qhov sib ntxiv ntawm cov tshuaj tiv thaiv preheated 2 x sib tov (kub pib ntawm cDNA synthesis).Qhov no pab tiv thaiv kev sib koom ua ke ntawm cov molecules ntawm qhov kub thiab txias.Siv cov cuab yeej PCR ua kom yooj yim rau ntau qhov kev hloov pauv kub uas xav tau rau RT-PCR.

3. Txo cov kab mob DNA genomic:

Ib qho teeb meem nyuaj nrog RT-PCR yog tias RNA paug DNA genomic.Kev siv txoj kev sib cais RNA zoo dua, xws li Trizol Reagent, txo cov kab mob DNA sib kis hauv RNA npaj.Txhawm rau zam cov khoom tsim los ntawm genomic DNA, RNA tuaj yeem kho nrog qib DnasⅠ txhawm rau tshem tawm cov kab mob DNA ua ntej rov qab hloov pauv.Cov qauv raug khaws cia ntawm 65 ℃ hauv 2.0mM EDTA rau 10 feeb kom tshem tawm DNaseⅠ kev zom.EDTA chelates magnesium ions los tiv thaiv magnesium ion dependent RNA hydrolysis uas tshwm sim ntawm qhov kub thiab txias.

Txhawm rau cais cov cDNA amplified los ntawm genome DNA amplification khoom, primers uas anneal cais nrog exon cais tau tsim.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.Ib qho kev sim tswj tsis tau thim rov qab kuj tseem ua rau txhua tus qauv RNA los txiav txim seb puas yog ib feem ntawm cov genomic DNA lossis cDNA.PCR cov khoom tau txais thaum tsis muaj kev hloov pauv rov qab yog muab los ntawm genome.

Yam khoom

RT-PCR Ib qho yooj yim^ᵀᴹKuv (Ib kauj ruam)

-Cov khoom siv ib-kauj ruam ua rau kev thim rov qab thiab PCR yuav tsum tau nqa tawm hauv tib lub raj.Nws tsuas yog xav tau ntxiv cov qauv RNA, tshwj xeeb PCR primers thiab RNase-Free ddH₂O.

-Real-time quantitative tsom xam ntawm RNA tuaj yeem ua tiav sai thiab raug.

-Cov khoom siv siv qhov tshwj xeeb Foregene thim rov qab reagent thiab Foregene HotStar Taq DNA Polymerase ua ke nrog cov tshuaj tiv thaiv tshwj xeeb txhawm rau txhim kho kev ua kom zoo dua qub thiab qhov tshwj xeeb ntawm cov tshuaj tiv thaiv.

-Cov tshuaj tiv thaiv zoo tshaj plaws ua rau cov tshuaj tiv thaiv muaj kev tshawb pom ntau dua, muaj zog thermal stability, thiab kam rau siab dua.

RT Yooj Yim II (Nrog GDNase) Master Premix Rau Thawj-Strand CDNA Synthesis Rau Lub Sijhawm PCR Nrog GDNase

-Kev muaj peev xwm tshem tawm gDNA, uas tuaj yeem tshem gDNA hauv cov qauv hauv 2 feeb.

-Txoj kev rov qab ua haujlwm tau zoo, nws tsuas yog siv sijhawm 15 feeb los ua kom tiav cov synthesis ntawm thawj strand cDNA.

-Cov qauv tsis yooj yim: cov qauv nrog cov ntsiab lus GC siab thiab cov qauv theem nrab kuj tuaj yeem thim rov qab nrog kev ua haujlwm siab.

-High-rhiab heev rov qab transcription system, pg-theem templates kuj tau txais high-zoo cDNA.

-Qhov kev hloov pauv rov qab muaj qhov thermal stability siab, qhov pom kev zoo ntawm qhov ntsuas kub yog 42 ℃, thiab nws tseem muaj qhov zoo rov qab ua haujlwm ntawm 50 ℃.