Txhua leej txhua tus tab tom tham txog lub hauv paus ntsiab lus ntawm qRT-PCR kev sim, kev tsim qauv, kev txhais lus, thiab lwm yam, tab sis kuv xav tias kuv yuav tsum qhia rau koj txog kev sim ua haujlwm ntawm qRT-PCR.Nws yog me me, tab sis nws yog hais txog cov txiaj ntsig.

Ua ntej ua qRT-PCR, peb yuav tsum muaj kev nkag siab meej txog peb tus kheej RNA thiab cov txheej txheem ua haujlwm.Tom qab tag nrho, peb cov kev siv zog yog tsom kom tau txais txiaj ntsig, tsis yog kev xyaum xwb.Yog li ua ntej ua qRT-PCR, peb yuav tsum txiav txim siab txog cov teeb meem hauv qab no (qee qhov tsuas yog siv tau rau SYBR).

1 Koj puas paub tseeb tias koj RNA tsis degraded?

NanoDrop 2000 tsuas tuaj yeem kuaj xyuas qhov concentration thiab purity ntawm RNA, tab sis tsis tuaj yeem kuaj xyuas qhov ncaj ncees ntawm RNA.

Tus nqi RNA (RNA Intestity Number) tuaj yeem cuam tshuam qhov kev ncaj ncees ntawm RNA, uas tau kuaj pom los ntawm Agilent 2100 Bioanalyzer system.

Daim duab schematic ntawm RIN qhov tseem ceeb rau cov qauv RNA sib txawv (eukaryotes)

Txawm li cas los xij, cov chaw kuaj mob feem ntau tsis muaj Agilent 2100 Bioanalyzer.Hauv qhov no, peb tuaj yeem tshawb xyuas los ntawm formaldehyde gel, tab sis qhov yuav tsum tau muaj rau tag nrho cov nyiaj RNA yog siab, yog li txoj kev ceev tshaj plaws yog siv cov gel electrophoresis zoo tib yam.Nws yuav tsum tau nyob rau hauv ib puag ncig tsis muaj nuclease, yog li nws yuav tsum tau yaug lub electrophoresis tank, sol fwj, gel bracket thiab zuag nrog dej DEPC.Lub agarose kuj tseem tsis muaj nuclease (tsuas yog nws tau qhib tshiab), thiab Loading Buffer yuav tsum tau qhib tshiab kom ntau li ntau tau, nrog 1.2% gel.

Nco ntsoov tias cov gel yuav tsum tau yaj tag, txwv tsis pub nws yuav ua rau tsis sib xws, raws li qhia hauv tus qauv 9 hauv daim duab.Yog tias qhov hluav taws xob siab dhau los yog khiav mus ntev dhau lawm yuav ua rau muaj cua sov thiab ua rau RNA degradation, yog li qhov voltage thiab lub sij hawm yuav tsum tau tswj kom tsim nyog.Tsis tas li ntawd, gel khiav kuj tseem tuaj yeem txiav txim siab ntxiv seb puas muaj DNA seem hauv cov qauv, thiab saib seb puas muaj ntau cov hlua khaws cia hauv qhov chaw xa khoom zoo.

Daim duab.Gel electrophoresis nrhiav pom ntawm RNA

2 Koj puas paub tseeb txog qhov concentration ntawm koj cDNA?

Cov kev paub ntawm cov kwv tij loj hauv chav kuaj yog tias cDNA ntawm 20 ul system tau los ntawm txhua qhov kev hloov pauv ncaj qha diluted 20X, thaum cov viv ncaus tom qab kws kho mob tau diluted 10X.Kuv feem ntau nyob ntawm qhov xwm txheej.Vim tias qhov zoo ntawm RNA hais los ntawm txhua tus neeg sib txawv, theem ntawm kev thim rov qab kuj txawv, thiab cov tshuab thim rov qab yuav tsis ruaj khov.

Yog li txhua zaus kuv tau txais qhov hloov pauv cDNA, kuv yuav xub dilute nws li 3 zaug, thiab tom qab ntawd siv cov noob hauv tsev los ua RT-PCR, cov voj voog feem ntau yog 25 lub voj voog, txhawm rau txheeb xyuas qhov tshwj xeeb, thiab tom qab ntawd txiav txim siab qhov kawg dilution.

3 Koj puas paub tseeb tias koj cov primers yooj yim siv?

Nws tuaj yeem dhau qhov nkhaus ntawm qRT-PCR, tab sis qhov no tseem raug nqi nyiaj.Rau cov chaw soj nstuam uas tsis muaj nyiaj ntau, thaum lawv tau txais cov primers ntau, lawv tuaj yeem siv RT-PCR zoo tib yam los saib seb nws puas yog ib pawg thiab txheeb xyuas qhov tshwj xeeb ntawm cov primers.Yog hais tias lub chaw soj nstuam tsis luv ntawm cov nyiaj, qhov tshwj xeeb ntawm tag nrho cov primers tuaj yeem raug txheeb xyuas ib zaug los ntawm qhov nkhaus melting.

4 Koj puas paub tseeb tias koj qhov kev sim ua kom haum?

SYBR yuav tsum muaj kev tiv thaiv los ntawm lub teeb muaj zog, yog li sim tua lub teeb hluav taws xob thaum ntxiv SYBR reagent, thiab tsuas yog yuav tsum siv lub teeb ci kom tiav.

Khaws SYBR ntawm 4 ° C.Thaum siv, maj mam invert up thiab down kom sib tov zoo kom tsis txhob foaming, thiab tsis txhob vortex vigorously.

Qee cov viv ncaus nyiam kos cov cim rau ntawm PCR lub rooj tsavxwm vim kev ntshai ntawm kev sib xyaw cov qauv, uas tsis yog.Vim tias koj cov cim muaj feem cuam tshuam rau kev sau cov cim fluorescent, feem ntau kuv xav kom cov menyuam yaus siv phau ntawv sim los pab hauv kev nco, raws li qhia hauv qab no.

Daim duab.qRT-PCR qauv loading daim duab

5 Koj puas paub tseeb tias koj ua qhov yog?

Nco ntsoov hnav hnab looj tes, hnav hnab looj tes, hnav hnab looj tes, thiab hais tej yam tseem ceeb peb zaug.

Txhawm rau txo qhov raug SYBR rau lub teeb, kuv tus kheej nyiam ntxiv cov qauv ua ntej, raws li qhia hauv daim duab hauv qab no.Raws li kev paub dhau los, qhov sib ntxiv ntawm ib qho me me ntawm cov qauv zoo li yuav ua rau cov qauv tsis raug.Yog li ntawd, txhawm rau txo qhov kev ua yuam kev los ntawm kev ntxiv me me ntawm cov qauv, kuv feem ntau muab ob npaug rau tus qauv dua, thiab muab ob npaug rau tus nqi thaum ntxiv cov qauv kom txo cov H2O2 ntxiv.

Daim duab.Schematic daim duab ntawm qRT-PCR loading

Tom qab ntawd teeb tsa qRT-PCR system raws li hauv qab no.

Daim duab.qRT-PCR system npaj daim duab

CEEB TOOM: Cov txheej txheem configuration yuav tsum tau ua rau dej khov.

Tom qab ntxiv cov qauv, muab tshuaj txhuam pob tshab sealing zaj duab xis.Sim tsis txhob kov lub pob tshab sealing zaj duab xis nrog koj txhais tes, tsuas yog ua haujlwm los ntawm qhov chaw ntawm ob sab ntawm zaj duab xis.Vim tias cov ntiv tes kuj tseem cuam tshuam rau kev sau cov cim fluorescent.Tom qab ntawd siv lub centrifuge kom ceev centrifuge rau 10 s ntawm qhov ceev kom tsis txhob muaj cov qauv los ntawm dai rau ntawm phab ntsa.

Yam khoom:

Cell Direct RT-qPCR cov khoom siv

RT Easy II