RT-qPCR kev sim suav nrog RNA rho tawm thiab kev soj ntsuam zoo, rov qab sau thiab qPCR peb kauj ruam, txhua kauj ruam muaj kev ceev faj ntau, peb yuav qhia kom meej hauv qab no.

Ⅰ.Kev soj ntsuam zoo RNA

Hauv kev sim RT-qPCR, tom qab ua tiav ntawm RNA rho tawm, qhov zoo ntawm RNA yuav tsum tau soj ntsuam, thiab kev sim ua raws li tsuas yog ua tau tom qab nws tsim nyog.Cov kev ntsuam xyuas muaj xws li spectrophotometer, Agilent gel electrophoresis, Agilent 2100 tsom xam, uas feem ntau siv spectrophotometer thiab agarose gel electrophoresis txoj kev tshawb nrhiav.Nws yuav tsum tau muab sau tseg tias ob txoj kev no yuav tsum tau siv ua ke los ua kom tiav qhov kev tshawb pom thiab kev tsom xam ntawm RNA concentration, purity thiab kev ncaj ncees, thiaj li ua kom ntseeg tau qhov zoo ntawm RNA.

Related RNA Isolation Kit: 

Cell Total RNA Isolation Kit

Ua kom huv thiab zoo tag nrho RNA tuaj yeem tau txais los ntawm ntau lub hlwb hauv 11min.

Tsiaj Tag Nrho RNA Isolation Kit

Ceev ceev thiab ua tau zoo extract high-purity thiab high-zoo tag nrho RNA los ntawm ntau yam tsiaj cov ntaub so ntswg.

Spectrophotometer:

Lub spectrophotometer feem ntau yog siv los txiav txim siab qhov concentration thiab purity ntawm RNA, tab sis nws tsis tuaj yeem txheeb xyuas qhov tseeb ntawm RNA thiab genomic residue.Ntawm lawv, A260/280 thiab A260/230 yog qhov tseem ceeb rau kev tshawb nrhiav RNA purity, thiab RNA purity tuaj yeem kuaj pom raws li qhov hloov pauv ntawm lawv cov txiaj ntsig:

1. 1.9< A260/280< 2.1, qhia tias RNA purity zoo;A260/280<1.9, qhia tias tej zaum yuav muaj cov protein seem hauv RNA;A260/280> 2.1, qhia tau ib nrab degradation ntawm RNA, uas tuaj yeem paub tseeb ntxiv los ntawm agarose gel electrophoresis.

2. 2.0< A260/230< 2.2, qhia tias RNA purity zoo;A260/230< 2.0, qhia tias tej zaum yuav muaj cov seem ntawm cov organic reagents hauv RNA, xws li phenols, ethanol lossis qab zib.

Agarose gel electrophoresis:

Agarose gel electrophoresis assay tuaj yeem txheeb xyuas RNA kev ncaj ncees, genome thiab cov protein seem, tab sis tsis tuaj yeem txheeb xyuas qhov tseeb ntawm RNA lossis kuaj pom cov seem ntawm cov organic reagents.Siv eukaryotic RNA templates piv txwv li:

1. RNA raug rau agarose gel electrophoresis.Yog tias tsuas muaj peb pawg ntawm 28sRNA, 18sRNA thiab 5.8sRNA ntawm daim ntawv qhia gel, nws qhia tau tias cov RNA rho tawm tsis zoo.Yog tias muaj qhov tshwm sim rub tawm, nws qhia tau tias ib nrab degradation ntawm RNA.

2. Yog tias muaj ib qho ci ci ntawm lub qhov kua nplaum thiab 28sRNA band, tej zaum yuav muaj genomic DNA residue.

3. Yog hais tias cov bands tshwm nyob rau hauv lub qhov kua nplaum, nws qhia tau hais tias tej zaum yuav muaj residues ntawm cov protein thiab lwm yam macromolecular tshuaj.

Ⅱ. Rov qab transcription

Tom qab RNA rho tawm tiav lawm, nws yuav tsum tau thim rov qab rau hauv cDNA rau kev sim tom ntej, yog li cov kauj ruam thim rov qab yog qhov tseem ceeb.Reverse transcription yuav raug qhia los ntawm kev xaiv ntawm thim rov qab transcriptase thiab primer:

Reverse transcriptase xaiv:

Cov ntaub ntawv rov qab raug suav nrog AMV RTase thiab MMLV RTase.RNase H ntawm AMV RTase muaj cov haujlwm muaj zog, luv synthesis ntev, qis synthesis tus nqi thiab zoo thermal stability (42 ~ 55 ℃).RNase H kev ua ntawm MMLV RTase tsis muaj zog, qhov sib xyaw ua ke ntev ntev, qhov sib xyaw ua ke yog siab, thiab thermal stability tsis zoo (37 ~ 42 ℃).

Vim tias RNase H enzyme muaj lub luag haujlwm ntawm degrading RNA template, MMLV uas tsis muaj zog RNase H kev ua haujlwm yuav tsum tau xaiv zoo dua thaum rov qab hloov dua siab tshiab, thiab tom qab tom qab cov noob caj noob ces, thermal stability ntawm MMLV tau mus txog qhov zoo leap.Noj ForegeneForeasy Reverse Transcriptase (M-MLV rau kev hloov pauv rov qab) Piv txwv li, nws yog ib qho kev rov qab transcriptase tshiab uas tau qhia hauv E. coli engineered cov kab mob uas siv cov cuab yeej siv tshuaj genetic recombination.Nws yog ib qho recombinant DNA polymerase uas ua ke cov DNA sib txuas los ntawm ib leeg-stranded RNA, DNA, lossis RNA: DNA hybrid.Nws tsis muaj RNase H kev ua haujlwm, muaj zog ruaj khov, muaj zog RNA affinity, thiab siab nrhiav kom tau rhiab heev.

 

Foreasy Reverse Transcriptase (M-MLV rau kev hloov pauv rov qab)

Kev xaiv ntawm primer:

Feem ntau RT primers poob rau hauv peb pawg: oligo dT, random primers, thiab gene-specific primers.Xaiv cov primers tsim nyog rau kev siv raws li kev sim sib txawv.

1. Yog hais tias tus qauv yog los ntawm eukaryotic keeb kwm thiab lub lig cDNA yog siv rau niaj hnub PCR amplification, Oligo (dT) yog pom zoo;Yog tias qhov kev sim tom ntej no tsuas yog siv rau qPCR, Oligo (dT) raug pom zoo kom muab sib xyaw nrog random primers los txhim kho qhov ua tau zoo ntawm kev thim rov qab.

2. Yog tias tus qauv yog los ntawm prokaryotes, Random Primers lossis gene tshwj xeeb primers yuav tsum raug xaiv rau kev hloov pauv rov qab.

Ⅲ.qPCR

Fluorescence kom muaj nuj nqis yog tsim los ntawm kev xaiv ntau txoj hauv kev, primer tsim qauv, ROX xaiv, cov tshuaj tiv thaiv kev teeb tsa thiab cov teeb meem tshuaj tiv thaiv, thiab lwm yam.

Kev xaiv ntau txoj hauv kev:

Cov txheej txheem Quantitative muab faib ua cov txheej txheem txheeb ze thiab cov txheej txheem ntau yam.Kev txheeb xyuas cov txheeb ze tuaj yeem siv los txheeb xyuas cov txiaj ntsig ntawm qee txoj kev kho mob ntawm cov noob qhia, txheeb xyuas qhov sib txawv ntawm cov noob qhia ntawm lub sijhawm sib txawv thiab sib piv qhov sib txawv ntawm cov noob qhia hauv cov ntaub so ntswg sib txawv.Kev txheeb xyuas qhov tseeb tuaj yeem kuaj pom tus nqi nucleic acid hauv tus kab mob thiab lwm yam.Thaum ua cov kev sim, peb yuav tsum xaiv cov qauv tsim nyog raws li peb tus kheej qhov kev sim.

Primer tsim qauv:

Tus tsim ntawm primer rau qPCR yog ncaj qha cuam tshuam rau lub amplification efficiency thiab cov khoom specification.Yog li ntawd, tsim nyog tsim cov primers zoo yog thawj kauj ruam ntawm kev ua tiav qPCR.Hauv kev tsim cov primer, cov hauv paus ntsiab lus hauv qab no yuav tsum tau them sai sai rau thaum ua tau raws li lub hauv paus ntsiab lus ntawm kev tsim primer:

1. Qhov ntev ntawm lub hom phiaj fragment yog tswj ntawm 100 thiab 300 bp;

2. Cross-exon tsim kom tsis txhob cuam tshuam ntawm genomic DNA;

3. Cov tsim primers yuav tsum tau sim rau amplification efficiency, thiab tsuas yog thaum lub amplification efficiency ncav cuag tus qauv (90-110%) lawv yuav siv tau rau ntau yam kev sim;

4. Primer concentration feem ntau yog optimized ntawm 0.1uM thiab 1.0uM.

Kev xaiv ntawmROX:

Nyob rau hauv tus txheej txheem ntawm cov tshuaj tiv thaiv kom muaj nuj nqis, ROX tuaj yeem kho qhov sib txawv ntawm txoj kev kho qhov muag, qhov yuam kev pipetting lossis ntim qhov sib txawv los ntawm evaporation thiab condensation uniformly, txhim kho qhov rov ua dua ntawm cov txiaj ntsig.Txawm li cas los xij, nws yuav tsum tau muab sau tseg tias kev xaiv ntawm ROX muaj feem xyuam nrog cov cuab yeej.Yog tias qPCR ntsuas muaj qhov ua haujlwm ntawm kev kho qhov sib txawv ntawm qhov, nws tsis tas yuav ntxiv ROX;txwv tsis pub, nws yuav tsum tau ntxiv ROX kho.Cov neeg koom tes me me hauv kev yuav cov tshuaj reagents yuav tsum yog raws li cov cuab yeej siv los xaiv qhov tseeb ROX, tsis txhob ua yuam kev tom qab.

Kev npaj cov tshuaj tiv thaiv kab mob:

Cov tshuaj tiv thaiv ntawm 20ul thiab 50ul yog nyiam.Cov teeb meem hauv qab no yuav tsum tau them sai sai rau thaum lub kaw lus raug tsim:

1. Cov txheej txheem tshuaj tiv thaiv yuav tsum tau npaj los ntawm qhov cua hauv lub workbench ultra-clean, ddH tshiab₂O yog siv rau txhua qhov kev sim;

2. Txhua qhov kev sim yuav tsum tau npaj NTC los xyuas seb puas muaj cov pa phem hauv lub cev, thiab txhua khub ntawm primers yuav tsum tau ua NTC thaum npaj lub kaw lus;

3. Txhawm rau txheeb xyuas seb puas muaj gDNA residue hauv RNA template, NRT tuaj yeem npaj rau txhua tus qauv rau kev kuaj pom;

4. Thaum npaj cov txheej txheem, nws raug nquahu kom ua yam tsawg kawg 3 kev rov ua dua rau ib qho qauv;

5. Thaum lub template yog cDNA, nws raug nquahu kom dilute 5-10 zaug kom txo tau cov nyhuv inhibition ntawm qhov rov qab transcription system ntawm qPCR kev sim.Nws yog qhov zoo dua los tshawb xyuas cov qauv kom muaj nuj nqis los ntawm gradient, kom tus nqi CT poob ntawm 20-30;

6. Txiav txim siab tus naj npawb ntawm cov tshuaj tiv thaiv, nce 5-10% raws li tus naj npawb ntawm cov tshuaj tiv thaiv, thiab suav cov naj npawb ntawm qhov ntim;

7, lub kaw lus tau npaj siv lub hauv paus ntsiab lus premix, sib tov tom qab centrifugation thiab xyuas kom tsis muaj npuas;

8, Raws li qhov ua tau los xaiv cov khoom siv txhawb nqa.

Yam khoom RT-qPCR