FastKing Ib Kauj Ruam RT-PCR Cov Khoom

A-1 RNA tau ua tsis zoo

—— Ua kom huv RNA zoo uas tsis muaj kev kis kab mob. Cov khoom siv los ntawm RNA uas tau muab rho tawm yuav tsum yog tshiab li sai tau los tiv thaiv RNA kev puas tsuaj. Txheeb xyuas RNA kev ncaj ncees ntawm denatured gel ua ntej RT cov tshuaj tiv thaiv. Tom qab RNA rho tawm, nws yuav tsum khaws cia hauv 100% formamide. Yog tias siv RNase inhibitor, qhov ntsuas cua sov yuav tsum yog <45 ° C, thiab pH yuav tsum tsawg dua 8.0, txwv tsis pub tus inhibitor yuav tso tag nrho RNase. Ntxiv mus, RNase inhibitor yuav tsum tau ntxiv hauv cov kev daws teeb meem uas muaj ≥ 0.8 mM DTT.

A-2 RNA muaj cov tshuaj tiv thaiv kab mob sib kis

——Resse transcription inhibitors suav nrog SDS, EDTA, glycerol, sodium pyrophosphate, spermidine, formamide, guanidine ntsev, thiab lwm yam. Ntxuav RNA nag lossis daus nrog 70% (v/v) ethanol tshem tawm cov tshuaj tiv thaiv.

A-3 Kev txhuam tsis txaus ntawm cov primers siv rau kev sib sau thawj cov hlua ntawm cDNA

—— Txiav txim siab tias qhov kub ua kom sov haum rau cov txheej txheem siv hauv kev sim. Rau qhov hexamers tsis raug, nws raug nquahu kom khaws qhov kub ntawm 25 ° C rau 10 feeb ua ntej mus txog qhov ntsuas kub. Rau cov noob tshwj xeeb (GSP), sim lwm GSP, lossis hloov mus rau oligo (dT) lossis random hexamer.

A-4 Me me ntawm kev pib RNA

—— Ua kom tus lej RNA ntau ntxiv. Rau cov qauv RNA tsawg dua 50 ng, 0.1 μg txog 0.5 μg acetyl BSA tuaj yeem siv hauv thawj txoj hlua cDNA synthesis

A-5 Lub hom phiaj ua ntu zus tsis tau hais tawm hauv cov ntaub so ntswg txheeb xyuas.

—— Sim lwm cov nqaij.

A-6 PCR cov tshuaj tiv thaiv tsis ua

—— Rau ob kauj ruam RT-PCR, cDNA tus qauv hauv PCR cov kauj ruam tsis tuaj yeem tshaj li 1/5 ntawm cov tshuaj tiv thaiv ntim.

A-1 Tsis-tshwj xeeb annealing ntawm primers thiab qauv

——Qhov 3'-kawg ntawm cov txheej txheem yuav tsum tsis muaj 2-3 dG lossis dC. Siv Gene-tshwj xeeb cov txheej txheem hauv thawj cov kab ke sib txuas ua ke siv cov primers random lossis oligo (dT). Siv qhov ntsuas kub kom siab dua hauv thawj ob peb mus, thiab tom qab ntawd txo qhov kub kom txias. Siv kub-pib Taq DNA polymerase rau PCR txhawm rau txhim kho qhov tshwj xeeb ntawm cov tshuaj tiv thaiv.

A-2 Kev tsim txom tsis zoo ntawm cov noob tshwj xeeb

—— Ua raws tib lub hauv paus ntsiab lus rau kev tsim qauv tsim ua ntej.

A-3 RNA paug nrog genomic DNA

—— Kho RNA nrog PCR-qib DNase I. Teeb tsa cov tshuaj tiv thaiv yam tsis thim rov qab los txheeb xyuas DNA sib kis.

A-4 Kev tsim ntawm primer dimer

—— Tsim cov txheej txheem yam tsis muaj ntu sib ntxiv ntawm 3 'kawg.

A-5 Mg siab heev²⁺ siab

--- Optimize Mg²⁺ concentration rau txhua tus qauv thiab ua ke ua ntej

A-6 Sib kis nrog DNA txawv teb chaws

—— Siv cov lus qhia tiv taus aerosol thiab UDG enzymes.

A-1 Cov ntsiab lus ntawm thawj cov khoom lag luam yog siab dhau

——Rescece tus nqi ntawm thawj cov khoom lag luam hauv cov txheej txheem tshuaj PCR ib txwm muaj.

A-2 Tus nqi loj heev hauv PCR cov tshuaj tiv thaiv

--- Txo cov tswv yim primer.

A-3 Ntau dhau mus

—— Ua kom zoo dua PCR cov kev mob tshwm sim thiab txo tus naj npawb ntawm PCR.

A-4 Qhov kub tsis txias ntau dhau

—— Ua kom sov qhov kub kom tiv thaiv qhov tsis yog qhov pib tshwj xeeb thiab txuas ntxiv.

A-5 Tsis yog qhov tshwj xeeb nthuav dav ntawm oligonucleotide tawg ua los ntawm DNase kev puas tsuaj ntawm DNA--Txheeb cais RNA zoo kom tiv thaiv DNA tsis zoo.

RT-PCR yog thim rov qab sau RNA rau hauv cDNA, thiab tom qab ntawd siv cov thim rov qab sau cDNA ua tus qauv rau PCR cov tshuaj tiv thaiv kom nthuav dav lub hom phiaj tawg. Xaiv ob qho kev xaiv ua piv txwv, Oligo dT thiab cov noob tshwj xeeb ua ntej raws li qhov xwm txheej tshwj xeeb ntawm kev sim. Txhua qhov txheej txheem saum toj no tuaj yeem siv rau luv eukaryotic cell mRNA yam tsis muaj cov qauv plaub hau.

Random primer: Tsim nyog rau RNA ntev nrog cov qauv plaub hau, ntxiv rau txhua yam RNA xws li rRNA, mRNA, tRNA, thiab lwm yam Lawv feem ntau yog siv rau RT-PCR cov tshuaj tiv thaiv ntawm ib tus qauv.

Oligo dT: Haum rau RNA nrog PolyA tailing (prokaryotic RNA, eukaryotic Oligo dT rRNA thiab tRNA tsis muaj PolyA tails). Vim tias Oligo dT tau khi rau PolyA tus tw, qhov ua tau zoo ntawm RNA cov qauv yuav tsum tau ua kom siab, thiab txawm tias qhov me me ntawm kev ua kom puas yuav txo qis qhov ua kom ntev cDNA synthesis.

Gene-specific primer: Ua tiav rau cov qauv ua ntu zus, tsim nyog rau cov xwm txheej uas paub lub hom phiaj ua ntu zus.

Muaj ob txoj hauv kev:

1. Cov txheej txheem siv sab hauv: Hauv txoj kev xav, cDNA yog DNA cov khoom tawg ntawm qhov sib txawv qhov ntev, yog li qhov tshwm sim ntawm electrophoresis yog smear. Yog tias RNA nplua nuj tsawg, tsis muaj cov khoom yuav qhia hauv electrophoresis, tab sis qhov no tsis txhais tau tias tsis muaj cov khoom lag luam yuav nthuav dav los ntawm PCR. Feem ntau, kev siv sab hauv tuaj yeem siv los txheeb xyuas cDNA. Yog tias cov ntaub ntawv siv sab hauv tau muaj txiaj ntsig, qhov zoo ntawm cDNA tuaj yeem lav tau zoo (hauv ob peb kis, yog tias lub hom phiaj tawg ua ntu zus ntev dhau los, yuav muaj qhov tshwj xeeb).

2. Yog tias muaj kev paub cov noob tau nthuav dav los ntawm tus qauv no, nws tuaj yeem txheeb xyuas tau los ntawm cov thawj ntawm cov noob no. Kev nthuav dav ntawm kev siv sab hauv tsis tas yuav txhais tau tias tsis muaj teeb meem nrog cDNA. Vim tias cov ntaub ntawv siv sab hauv muaj qib siab hauv cDNA, nws yog qhov yooj yim kom nthuav dav. Yog tias cDNA qee yam tsis zoo rau ntau qhov laj thawj, los ntawm kev xav txog qhov tshwm sim, PCR cov txiaj ntsig ntawm cov noob qis uas muaj lub hom phiaj yuav raug cuam tshuam loj heev. Txawm hais tias kev siv sab hauv tseem muaj ntau hauv kev nplua nuj, kev nthuav dav yuav zaum tsis cuam tshuam.

Ib nrab degrade ntawm RNA. Txheeb xyuas qhov ncaj ncees thiab ua kom huv ntawm RNA

RNA cov ntsiab lus ntawm cov tsiaj sib txawv tuaj yeem sib txawv, tab sis feem ntau, rho tawm tag nrho RNA yuav tsum muaj ob qhov tseeb 28S thiab 18S bands hauv gel electrophoresis, thiab qhov ci ntawm cov qhab qub yuav tsum yog ob zaug siab dua li qhov tom kawg. Cov qhab 5S qhia tias RNA tau poob qis, thiab nws qhov ci ci yog qhov sib piv rau qib kev puas tsuaj. Kev ua tiav kev nthuav dav ntawm kev siv sab hauv tsis txhais tau tias tsis muaj teeb meem nrog RNA, vim tias kev siv sab hauv yog qhov muaj ntau, RNA tuaj yeem nthuav dav kom ntev li ntev tau qhov kev puas tsuaj tsis hnyav. Cov OD₂₆₀/OS₂₈₀piv ntawm RNA ntshiab ntsuas los ntawm spectrophotometer yuav tsum nyob nruab nrab ntawm 1.9 thiab 2.1. Ib qho me me ntawm cov protein tsis huv hauv RNA yuav txo qhov sib piv. Tsuav tus nqi tsis qis heev, RT yuav tsis cuam tshuam. Dab tsi tseem ceeb tshaj plaws rau RT yog RNA ncaj ncees.

Kev txuas ntxiv ntawm cov noob siv sab hauv tuaj yeem tsuas yog qhia tias RT tau ua tiav, tab sis nws tsis tas yuav cuam tshuam nrog qhov zoo ntawm cDNA strand. Vim tias cov ntaub ntawv siv sab hauv feem ntau me me hauv qhov loj thiab siab hauv kev nthuav qhia, lawv yooj yim dua kom ua tiav hauv kev thim rov qab. Txawm li cas los xij, qhov loj me thiab qhia tawm ntawm cov hom phiaj sib txawv ntawm cov noob mus rau noob. Qhov zoo cDNA tsis tuaj yeem txiav txim siab tsuas yog los ntawm kev siv sab hauv tshwj xeeb rau cov phiaj tawg ua haujlwm ntev dua 2 kb.

Qee qhov piv txwv muaj cov txheej txheem thib ob nyuaj, lossis muaj cov ntsiab lus nplua nuj GC, lossis muaj nuj nqis nrog kev nplua nuj tsawg. Hauv cov xwm txheej no, qhov tsim nyog thim rov qab transcriptase yuav tsum raug xaiv raws li qhov loj me ntawm cov phiaj tawg thiab cov qauv. Rau RNA cov qauv uas muaj GC cov ntsiab lus siab thiab cov qauv txheej txheem nyuaj, nws nyuaj rau qhib cov qauv thib ob ntawm qhov kub tsawg, lossis nrog cov thim rov qab zoo ib yam. Txog cov qauv no, Quant Reverse Transcriptase tuaj yeem xaiv tau, txij li nws rov qab ua cov ntawv sau tau zoo yog qhov zoo dua li ntawm M-MLV series thim rov qab transcriptase, uas tuaj yeem thim rov qab sau ntau yam RNA tus qauv kom zoo thiab sau RNA rau hauv cDNA thawj txoj hlua mus rau qhov siab tshaj plaws. Thaum siv cov khoom thim rov qab dav dav, 20 systeml system tuaj yeem ua haujlwm tau zoo thim rov qab sau 1 μg ntawm tag nrho RNA. Thov xyuam xim rau qhov peev xwm RT siab tshaj plaws ntawm cov khoom siv. Yog tias tus qauv raug ntxiv nyob rau hauv ntau dhau, thim rov qab cov ntawv yuav nyiam RNA nrog kev nplua nuj. Yog li ntawd, nws yog qhov zoo dua kom tsis txhob tshaj qhov muaj peev xwm tshaj plaws ntawm qhov system.

A-1 Txiav txim siab yog tias RNA puas ntsoog loj heev thiab yog tias RT ua tiav

Feem ntau, qhov laj thawj rau qhov ua tsis tiav ntawm kev siv sab hauv nthuav dav feem ntau tshwm sim los ntawm kev puas tsuaj loj RNA. Lwm qhov laj thawj yog thim rov qab cov ntawv sau tsis tiav. Kev siv sab hauv tsis tuaj yeem siv los ua tus qauv los txiav txim qhov zoo ntawm cDNA ib leeg, tab sis nws tuaj yeem siv los ua tus qauv los txiav txim seb kev thim rov qab ua tiav yog tsis muaj teeb meem ntawm RNA zoo. Qhov tseem ceeb tshaj plaws hauv cov txheej txheem thim rov qab yog kom tswj tau qhov kub thiab qhov ua haujlwm tsis tu ncua txhawm rau txhawm rau txhim kho cov tshuaj tiv thaiv kev ua haujlwm.

A-2 Txiav txim siab seb puas tseem ceeb rau kev nthuav dav sab hauv cov noob siv tau ntseeg tau thiab yog tias muaj teeb meem nrog cov tshuaj reagents siv hauv PCR.

Txog kev txheeb xyuas qhov txheeb ze, RNA yuav tsum suav ua ntej thim rov qab cov ntawv, uas tseem yuav tsum muaj nyob hauv ntau cov khoom siv thim rov qab, piv txwv li, ntsuas RNA cov tswv yim li 1 μg. Txij li qhov rov qab sau dua cDNA yog kev sib xyaw ua ke, suav nrog RNA, oligo dT, enzyme, dNTP, thiab txawm tias muaj DNA tsawg me ntsis, qhov sib txawv yuav tshwm sim, yog li nws tsis yooj yim sua kom muaj nuj nqis cDNA. Yog li ntawd, RNA kev suav yog tsim nyog. Xav txog qhov rov ua dua cov ntawv sau tseg tau zoo ib yam ntawm cov qauv sib txawv, tus nqi ntawm cDNA tau txais yuav tsum zoo ib yam, thiab kev tshuaj xyuas ntau tuaj yeem qhia qhov sib piv ntawm kev qhia qib ntawm cov noob sib txawv hauv tib tus lej ntawm RNA tag nrho. Thaum ua qhov txheeb ze fluorescence quantitative PCR, ntau qhov cDNA yuav tsis xav tau tom qab thim rov qab vim tias cov noob siv sab hauv tuaj yeem ua raws li kev siv.

Nws yog feem ntau cuam tshuam nrog cov noob, thiab thim rov qab cov lus ntawm qhov tawg ntev yog tsis ua tau rau feem ntau cov noob. Ua ntej, qhov ua tau zoo ntawm kev thim rov qab yog qis dua li ntawm PCR. Qhov thib ob, GC thaj av nplua nuj thiab qib thib ob ntawm ntau cov noob txwv ob qho tib si thim rov qab thiab PCR. Thaum kawg, kev ncaj ncees thiab ua kom tau zoo ntawm PCR nyuaj rau lav tib lub sijhawm. Hauv cov txheej txheem ntawm kev thim rov qab, tsis muaj leej twg tuaj yeem lav kom tau txais qhov tawg ntev rau cov ntawv theej tsawg, tshwj xeeb yog siv oligo dT. Raws li rau 5 'UTR nrog ntau GC, nws tseem nyuaj dua. Yog li ntawd, nws tseem yog ib txoj hauv kev tsim nyog rau thim rov qab cov ntawv sau nrog cov ntawv tsis sib xws, nrhiav qhov chaw tshem tawm ntuj hauv cov phiaj tawg, nthuav dav los ntawm ntu, thiab tom qab ntawd ua qhov txwv kev zom thiab ligation. Feem ntau, nws nyuaj rau ncaj qha nthuav cov tawg loj dua 2 kb, tab sis nws tsis yog ib txwm ua tsis tau: 1. Ua ntej tshaj plaws, lav qhov ncaj ncees ntawm RNA/mRNA, thiab TRIZOL rho tawm yog qhov nyiam. 2.M-MLV RT-PCR cov khoom siv tau ncaj qha. Ntev ncua sijhawm kho thiab nce tus lej hauv cov txheej txheem ua kom raug kom raug. Ib qho ntxiv, zes PCR tuaj yeem thov, lossis ua ib lossis ob qhov kev cuam tshuam ua ntej nrog qhov tsim nyog ncua sij hawm ncua thiab ncua sijhawm ua ntej li qub PCR kev nthuav dav, uas tuaj yeem pab txuas ntxiv qhov tawg. Them sai sai rau kev ncaj ncees ntawm polymerase. 3.Long Taq tuaj yeem siv hauv PCR kom tau txais txiaj ntsig zoo. 4.For cov ntaub ntawv qhia txog protein, siab ncaj polymerase yuav tsum tau siv.

Muaj ob hom kev thim rov qab muab los ntawm TIANGEN: Quant/King RTase thiab TIANScript M-MLV. Qhov sib txawv tseem ceeb ntawm lawv yog cov nqi ntawm cov qauv. Quant yog qhov txawv txav transcriptase, uas txawv los ntawm kev siv M-MLV feem ntau tau los ntawm Moloney murine leukemia virus. Quant yog qhov ua tau zoo dua tshiab thim rov qab transcriptase rov hais dua los ntawm engineering Escherichia coli. Quantity yog qhov tsim nyog rau kev nthuav dav 50 ng-2 μg ntawm RNA nrog kev ua haujlwm siab rov qab ua haujlwm thiab ua tau zoo. Piv nrog MMLV zoo ib yam lossis AMV, Quant tus yam ntxwv loj tshaj plaws yog tias nws muaj kev sib raug zoo nrog RNA tus qauv thiab tuaj yeem thim rov qab cov ntawv sau ua piv txwv yam tsis muaj qhov kub siab. Rau cov qauv uas muaj cov ntsiab lus GC siab dua, qhov rov ua haujlwm tau zoo dua. Txawm li cas los xij, daim ntawv thim rov qab no muaj RNase H cov haujlwm, uas tuaj yeem cuam tshuam qhov ntev ntawm cDNA cov khoom (haum rau <4.5 kb tus qauv). Txog kev rov sau dua ib txwm, TIANScript MMLV thim rov qab transcriptase tau pom zoo. Qhov RTase no yog hloov pauv enzyme nrog RNase H tsis muaj zog txaus, uas tsim nyog rau ntev (> 5 kb) cDNA synthesis.

Ib kauj ruam thim rov qab thiab PCR kev nthuav dav tau ua tiav hauv tib lub raj yam tsis tau qhib lub raj npog ntawm cDNA kev sib xyaw thiab ua kom nrov, uas yog qhov pab txo qis kev sib kis. Txij li txhua qhov piv txwv cDNA tau txais yog siv rau kev nthuav dav, qhov ua tau zoo dua, nrog qhov tsawg kawg ntawm 0.01 pg ntawm tag nrho RNA. Rau kev ua tiav ib kauj ruam RTPCR, cov noob tshwj xeeb rau cov noob feem ntau yog siv los pib cDNA kev sib xyaw. Cov txheej txheem ob kauj ruam, uas yog thim rov qab sau thiab PCR kev nthuav dav tau ua tiav hauv ob kauj ruam. Thawj zaug thim rov qab cov ntawv sau tau ua los ntawm tus qauv RNA kom tau txais cDNA, thiab qhov tau txais cDNA raug rau ib lossis ntau qhov sib txawv ntawm PCR cov tshuaj tiv thaiv. Txoj hauv kev ob kauj ruam tuaj yeem siv oligo (dT) lossis cov txheej txheem tsis sib xws los qhia kev sib txuas ntawm thawj kab ntawm cDNA, thiab tuaj yeem thim rov qab sau tag nrho cov ntaub ntawv mRNA los ntawm cov qauv tshwj xeeb.