1. Kev nkag siab pib
Nyob rau theem no, peb yuav tsum nkag siab qee lub tswv yim thiab cov lus siv, kom tsis txhob ua yuam kev ntawm peb cov neeg laus, xws li:
Q: Qhov txawv ntawm RT-PCR, qPCR, Real-time PCR, thiab real-time RT-PCR yog dab tsi?
Teb: RT-PCR yog thim rov qab PCR(reverse transcription PCR, RT-PCR), uas yog ib tug dav siv ntau yam ntawm polymerase saw cov tshuaj tiv thaiv (PCR).Hauv RT-PCR, ib qho RNA strand yog thim rov qab rau hauv DNA ntxiv, uas yog siv los ua tus qauv rau DNA amplification los ntawm PCR.
Real-time-PCR thiab qPCR(Quantitative Rea-ltime-PCR) yog tib yam, ob qho tib si yog PCR ntawm lub sijhawm tiag tiag, uas txhais tau hais tias txhua lub voj voog ntawm PCR muaj cov ntaub ntawv teev sijhawm tiag tiag, yog li tus naj npawb ntawm cov qauv pib tuaj yeem hloov kho qhov kev tshuaj xyuas meej.
Txawm hais tias ob qho tib si Real-time PCR (real-time fluorescent quantitative PCR) thiab Reverse transcription PCR (reverse transcription PCR) zoo li abbreviated li RT-PCR, lub thoob ntiaj teb convention yog: RT-PCR tshwj xeeb yog hais txog rov qab transcription.PCR, Real-time PCR feem ntau yog luv li qPCR (quantitative real-time PCR).
Thiab lub sijhawm tiag tiag RT-PCR (RT-qPCR), Nws yog qhov thim rov qab PCR ua ke nrog cov tshuab hluav taws xob ntau ntau: thawj zaug tau txais cDNA (RT) los ntawm RNA thim rov qab, thiab tom qab ntawd siv Real-time PCR rau kev txheeb xyuas ntau (qPCR).Feem ntau lub chaw soj nstuam ua RT-qPCR, uas yog, kev tshawb fawb ntawm RNA kev qhia down-regulation, yog li qPCR uas txhua leej txhua tus tham txog nyob rau hauv lub chaw soj nstuam tiag tiag yog hais txog RT-qPCR, tab sis tsis txhob hnov qab tias tseem muaj ntau ntau DNA kuaj nyob rau hauv cov ntaub ntawv kho mob.Kev tshuaj xyuas kom muaj nuj nqis, xws li kev kuaj pom tus kab mob siab B HBV.
Nqe Lus Nug: Tom qab nyeem ntau cov fluorescent quantitative PCR, vim li cas thiaj li yuav tsum tau ua kom muaj qhov sib txawv ntawm qhov sib txawv ntawm 80-300bp?
Teb: Qhov ntev ntawm txhua qhov gene sequence sib txawv, qee qhov yog ob peb kb, qee qhov ntau pua bp, tab sis peb tsuas yog xav kom cov khoom ntev yuav tsum yog 80-300bp thaum tsim cov primers, luv luv lossis ntev dhau tsis haum rau fluorescent quantitative PCR nrhiav kom tau.Cov khoom tawg yog luv dhau los ua qhov txawv ntawm cov primer-dimer.Qhov ntev ntawm primer-dimer yog hais txog 30-40bp, thiab nws yog ib qho nyuaj rau kev paub qhov txawv seb nws yog primer-dimer los yog ib yam khoom yog tias nws tsawg dua 80bp.Yog tias cov khoom tawg tau ntev dhau lawm, ntau tshaj 300bp, nws yuav yooj yim ua rau kev ua kom tsis muaj zog tsawg thiab tsis tuaj yeem kuaj pom tus nqi ntawm cov noob.
Piv txwv li, thaum koj suav pes tsawg tus neeg nyob hauv chav kawm, koj tsuas yog yuav tsum suav pes tsawg lub qhov ncauj xwb.Ib yam yog qhov tseeb thaum koj kuaj cov noob, koj tsuas yog yuav tsum tau kuaj xyuas qee yam kab ke ntawm cov noob los sawv cev Tag nrho cov kab ke yuav ua.Yog tias koj xav suav tib neeg, koj yuav tsum suav ob lub qhov ncauj thiab qhov ntswg, pob ntseg, thiab tsom iav, thiab nws yooj yim ua yuam kev.
Txhawm rau nthuav dav, hauv kev tshawb fawb lom neeg, muaj ntau qhov kev tshawb fawb los ntawm taw tes mus rau thaj chaw, vim tias cov noob kab lis kev cai ntawm txhua hom yog ntev heev, nws tsis tsim nyog thiab tsis tuaj yeem ntsuas tag nrho cov kab mob, xws li kab mob 16S sequencing, uas yog ua raws li cov kab mob sib xyaw ua ke ntawm cov kab mob Assays infer tus naj npawb ntawm qee cov kab mob.
Q: Ntev npaum li cas rau qPCR primer tsim?
Teb: Feem ntau hais lus, lub primer ntev yog hais txog 20-24bp, uas yog qhov zoo dua.Tau kawg, peb yuav tsum tau them sai sai rau TM tus nqi ntawm cov primer thaum tsim cov primer, vim qhov no muaj feem xyuam rau qhov zoo tshaj plaws annealing kub.Tom qab ntau qhov kev sim, nws tau raug pov thawj tias 60 ° C yog tus nqi TM zoo dua.Yog hais tias qhov kub ntawm annealing tsawg dhau lawm, nws yuav yooj yim ua rau tsis muaj qhov tshwj xeeb amplification.Yog hais tias qhov kub ntawm annealing siab dhau lawm, lub amplification efficiency yuav tsawg, lub ncov ntawm lub amplification nkhaus yuav pib tom qab, thiab CT tus nqi yuav ncua.
Q: Txoj kev zas xim txawv li cas los ntawm txoj kev sojntsuam?
Teb: Dye methodQee cov fluorescent dyes, xws li SYBR Green Ⅰ, PicoGreen, BEBO, thiab lwm yam, tsis tawm lub teeb los ntawm lawv tus kheej, tab sis yuav tawm fluorescence tom qab khi rau qhov me me ntawm ob-stranded DNA.Yog li ntawd, thaum pib ntawm PCR cov tshuaj tiv thaiv, lub tshuab tsis tuaj yeem ntes cov teeb liab fluorescent.Thaum cov tshuaj tiv thaiv mus txog rau theem annealing-extension, ob txoj hlua tau qhib, thiab ib txoj hlua tshiab yog tsim los ntawm DNA polymerase, thiab cov fluorescent molecule khi rau dsDNA me zawj.Raws li tus naj npawb ntawm PCR cycles nce, ntau thiab ntau dyes tau ua ke nrog ob-stranded DNA, thiab cov teeb liab fluorescent kuj tseem txhim kho tsis tu ncua.Txoj kev dye feem ntau yog siv hauv kev tshawb fawb tshawb fawb.
PS: Ceev faj thaum ua qhov kev sim, cov xim yuav tsum tau ua ke nrog tib neeg DNA, ceev faj kom tig mus rau hauv tus neeg fluorescent.
Dye method (sab laug) Probe method (sab xis)
PS: Ceev faj thaum ua qhov kev sim, cov xim yuav tsum tau ua ke nrog tib neeg DNA, ceev faj kom tig mus rau hauv tus neeg fluorescent.
SYBR Green Ⅰ khi rau qhov me me ntawm DNA
Txoj kev sojntsuamTaqman sojntsuam yog feem ntau siv hydrolysis sojntsuam.Muaj ib pab pawg fluorescent nyob rau ntawm 5' kawg ntawm qhov kev sojntsuam, feem ntau yog FAM, thiab qhov kev sojntsuam nws tus kheej yog qhov sib txuas ntxiv rau cov hom phiaj.Muaj ib pab pawg fluorescent quenching ntawm 3′ kawg.Raws li lub hauv paus ntsiab lus ntawm fluorescence resonance zog hloov (Förster resonance zog hloov, FRET), thaum lub reporter fluorescent pab pawg neeg (neeg pub fluorescent molecule) thiab quenching fluorescent pab pawg neeg (acceptor fluorescent molecule) zoo siab thaum lub spectra overlap thiab qhov deb heev ze (7-10nm), lub exacerbation ntawm lub molecule ntawm lub molecule tau txais. , thaum lub autofluorescence tsis muaj zog.Yog li ntawd, thaum pib ntawm PCR cov tshuaj tiv thaiv, thaum qhov kev sojntsuam yog dawb thiab tsis zoo nyob rau hauv lub cev, cov neeg sau xov xwm fluorescent pawg yuav tsis tawm fluorescence.Thaum annealing, primer thiab sojntsuam khi rau lub template.Thaum lub sijhawm txuas ntxiv, cov polymerase txuas ntxiv txuas ntxiv cov saw tshiab.DNA polymerase muaj 5′-3′ exonuclease kev ua.Thaum mus txog qhov kev sojntsuam, DNA polymerase yuav hydrolyze qhov kev sojntsuam los ntawm tus qauv, cais cov neeg sau xov xwm fluorescent los ntawm pawg quencher fluorescent, thiab tso tawm cov teeb liab fluorescent.Txij li thaum muaj kev sib raug zoo ib-rau-ib qho ntawm qhov kev sojntsuam thiab tus qauv, txoj kev sojntsuam yog qhov zoo tshaj rau txoj kev dye raws li qhov tseeb thiab qhov rhiab ntawm qhov kev xeem.Txoj kev sojntsuam feem ntau yog siv rau kev kuaj mob.
Q: Dab tsi yog qhov kom muaj nuj nqis?Relative Quantification yog dab tsi?
Teb: Kev txheeb xyuas qhov tseeb yog hais txog kev suav ntawm thawj tus lej luam ntawm tus qauv coj mus kuaj los ntawm qPCR, xws li muaj pes tsawg tus kab mob HBV nyob hauv 1ml ntawm cov ntshav.Cov txiaj ntsig tau los ntawm kev txheeb xyuas qhov txheeb ze yog qhov hloov pauv ntawm tus nqi ntawm cov hom phiaj hauv ib qho piv txwv tshwj xeeb txheeb ze rau lwm tus qauv siv, thiab cov gene qhia tau nce- tswj lossis txo qis.
Q: Puas yog tus nqi ntawm RNA rho tawm, thim rov qab kev ua haujlwm, thiab kev ua kom zoo dua qub puas cuam tshuam rau qhov kev sim?
Q: Puas yog cov qauv khaws cia, rho tawm reagents, thim rov qab cov ntaub ntawv reagents, thiab cov khoom siv hluav taws xob xa hluav taws xob puas cuam tshuam rau qhov kev sim?
Q: Txoj kev twg tuaj yeem kho cov ntaub ntawv sim?
Hais txog cov teeb meem no, peb yuav piav qhia rau lawv hauv cov ncauj lus kom ntxaws thiab qib siab hauv qab no.
2. Kev paub Advanced
Hais txog lub sijhawm tiag tiag fluorescent quantitative PCR, peb yuav tsum paub qhov tseeb tias ntau txhiab daim ntawv tshawb fawb tshawb fawb tau luam tawm txhua xyoo, ntawm cov fluorescent quantitative PCR thev naus laus zis tsis yog tsawg.
Yog tias tsis muaj tus qauv zoo los ntsuas qhov fluorescent quantitative PCR kev sim, cov txiaj ntsig yuav txawv.Rau tib lub noob ntawm tib hom, nrog rau tib txoj kev ua, cov txiaj ntsig tshawb pom kuj yuav sib txawv, thiab nws yuav nyuaj rau cov neeg latecomers rov ua cov txiaj ntsig qub.Koj Tsis muaj leej twg paub qhov twg yog qhov twg yog qhov tsis raug.
Puas yog qhov no txhais tau hais tias fluorescent quantitative PCR yog cov cuab yeej dag lossis cov cuab yeej siv tsis tau?Tsis yog, nws yog vim fluorescent quantitative PCR ntau rhiab thiab raug ntau dua, thiab kev ua haujlwm tsis raug me ntsis yuav ua rau cov txiaj ntsig tsis zoo.Ib qho kev poob me me yog ib txhiab mais deb.Tus sau tsab xov xwm yuav raug tsim txom ntau zaus los ntawm cov neeg tshuaj xyuas.Nyob rau tib lub sijhawm, cov neeg tshuaj xyuas ntawm phau ntawv xov xwm kuj nyuaj rau xaiv los ntawm cov txiaj ntsig kev sim sib txawv.
Txhua yam, taw qhia rau qhov tsis muaj kev pom zoo hauv kev sim PCR tiag tiag.Txog qhov kawg no, cov kws tshawb fawb laus hauv kev lag luam pib tsim cov qauv,xav kom cov neeg koom nrog muab qee qhov kev sim thiab cov ntaub ntawv ua tiav (nrog rau cov ntaub ntawv tsim nyog) hauv kab lus kom ua tau raws li cov qauv no.
Cov neeg soj ntsuam tuaj yeem txiav txim siab qhov zoo ntawm qhov kev sim los ntawm kev nyeem cov ntsiab lus no;Cov neeg nyeem yav tom ntej kuj tuaj yeem siv qhov no los rov ua qhov kev sim lossis txhim kho qhov kev sim.Tom qab ntawd cov kev sim tau txais nyob rau hauv txoj kev no yog tag nrho ntawm cov ntaub ntawv, zoo, thiab siv tau.
MIBBI (Cov ntaub ntawv tsawg kawg rau Kev Tshawb Fawb Txog Kev Kho Mob thiab Kev Kho Mob Biomedical -http://www.miv.org) tau los ua.MIBBI yog ib qhov project uas muab cov qauv rau kev sim.Nws yog luam tawm nyob rau hauv xwm.Txoj haujlwm no yog tsom mus rau ntau yam kev sim tshuaj lom neeg, suav nrog cell biology, Microarray, qPCR peb tab tom tham txog tam sim no, thiab lwm yam, thiab muab rau txhua hom kev sim thaum xa cov ntawv sau.Cov ntaub ntawv ntawd yuav tsum tau muab txhua lub sijhawm.
Hauv MIBBI qhov project, muaj ob nqe lus hais txog fluorescent quantitative PCR, uas yog:
· RDML (Real-Time PCR Data Markup Language) - cov lus qhia thiab qhia txog cov ntaub ntawv PCR tiag tiag;
· MIQE (Cov ntaub ntawv tsawg kawg nkaus rau Kev Tshaj Tawm Txog Quantitative Real-Time PCR Experiments) - Cov ntaub ntawv yam tsawg kawg nkaus rau kev tshaj tawm cov lus hais txog kev sim PCR ntawm lub sijhawm.
Ua ntej, cia peb tham txog RDML, cov lus qhia tshwj xeeb.
Yog tias tsis muaj tus qauv txhais rau txhua yam, nws tsis tuaj yeem txuas ntxiv kev sib tham, yog vim li cas qhov kev piav qhia ntawm cov ntsiab lus tseem ceeb heev hauv kev xeem.
Cov lus siv nyob rau hauv qhov kev sim fluorescent quantitative PCR suav nrog cov ntsiab lus hauv qab no.QIAGEN tau ua cov ntsiab lus zoo tshaj plaws rau peb.Cov nram qab no yog tag nrho qhuavkhoom .
Amplification nkhaus
Qhov kev nthuav dav nkhaus yog hais txog qhov nkhaus uas tau ua thaum lub sijhawm PCR txheej txheem, nrog rau lub voj voog tus lej raws li abscissa thiab lub sijhawm siv fluorescence tiag tiag thaum lub sijhawm cov tshuaj tiv thaiv raws li kev tswj hwm.
Qhov zoo tshaj plaws amplification nkhaus yuav tsum muaj cov yam ntxwv hauv qab no: lub hauv paus yog tiaj tus lossis qis me ntsis, thiab tsis muaj qhov pom tseeb nce siab;lub inflection point ntawm qhov nkhaus yog qhov tseeb, thiab txoj kab nqes ntawm lub exponential theem yog proportional rau lub amplification efficiency.Qhov ntau dua txoj kab nqes, qhov ua kom muaj zog ntau dua;tag nrho amplification nkhaus Qhov parallelism yog qhov zoo, qhia tias qhov kev ua kom zoo ntawm txhua lub raj zoo sib xws;lub exponential theem ntawm amplification nkhaus ntawm low-concentration qauv yog pom tseeb.
Baseline (zeeg)
Lub hauv paus yog lub suab nrov ntawm lub voj voog thaum ntxov, feem ntau ntsuas nruab nrab ntawm 3rd thiab 15th voj voog, vim hais tias tus nqi fluorescence nce los ntawm cov khoom amplification tsis tuaj yeem kuaj pom lub sijhawm no.Tus naj npawb ntawm cov voj voog siv los xam cov hauv paus hauv paus tuaj yeem sib txawv thiab yuav tsum tau txo qis yog tias siv cov template siab lossis yog qhov qhia theem ntawm cov hom phiaj siab.
Teem lub hauv paus yuav tsum tau saib cov ntaub ntawv fluorescence los ntawm linearity amplification nkhaus.Lub hauv paus txheej txheem yog teem kom qhov kev loj hlob ntawm lub amplification nkhaus pib nrog lub voj voog tus lej ntau dua li lub hauv paus voj voog saum toj kawg nkaus.Cov hauv paus ntsiab lus yuav tsum tau teeb tsa tus kheej rau txhua lub hom phiaj.Qhov nruab nrab fluorescence qhov tseem ceeb pom nyob rau hauv lub voj voog thaum ntxov yuav tsum tau muab rho tawm los ntawm cov txiaj ntsig fluorescence tau txais hauv cov khoom lag luam amplified.Qhov tseeb versions ntawm ntau yam Real-Time PCR software tso cai rau optimization ntawm lub hauv paus chaw rau ib tug neeg kuaj.
Thaum thawj ob peb lub voj voog ntawm PCR amplification cov tshuaj tiv thaiv, lub teeb liab fluorescence tsis hloov ntau.Mus cuag ib txoj kab ncaj nraim yog hu ua lub hauv paus, tab sis yog tias peb saib ze rau ntawm thawj ob peb lub voj voog, peb pom tias nyob rau hauv lub hauv paus yog dab tsi tshwm sim hauv daim duab hauv qab no.
Background Background hais txog
tus nqi tsis tshwj xeeb fluorescence hauv cov tshuaj tiv thaiv.Piv txwv li: tsis muaj fluorescence quenching;los yog ib tug loj tus naj npawb ntawm ob-stranded DNA templates vim yog siv SYBR Green.Cov keeb kwm yav dhau ntawm lub teeb liab raug muab tshem tawm los ntawm Real-Time PCR software algorithm.
Xov xwm teeb liab
Cov xov xwm ceeb toom yog hais txog cov teeb liab fluorescent tsim los ntawm SYBR Green lossis fluorescently sau npe raws li cov kev sojntsuam tshwj xeeb thaum lub sijhawm PCR.
Normalized Reporter Signal (RN)
RN yog hais txog kev siv fluorescence ntawm tus neeg sau xov xwm dye faib los ntawm kev siv fluorescence ntawm passive reference dye ntsuas ntawm txhua lub voj voog.
Passive Reference Dye
Hauv qee lub sijhawm PCRs,ROX fluorescent dye yog siv los ua ib qho kev siv sab hauv kom normalize lub teeb liab fluorescent.Nws kho qhov kev hloov pauv vim qhov tsis raug pipetting, txoj hauj lwm zoo, thiab fluorescence fluctuations ntawm qhov zoo ntawm qhov zoo.
fluorescence pib (threshold)
tau hloov kho saum toj no tus nqi keeb kwm yav dhau thiab tseem ceeb hauv qab tus nqi toj siab ntawm qhov amplification nkhaus.Nws yuav tsum dag nyob rau hauv cheeb tsam linear ntawm lub amplification nkhaus, sawv cev rau lub log-linear ntau ntawm PCR nrhiav pom.Cov theem pib yuav tsum tau teem rau hauv lub log-amplification nkhaus saib kom lub log-linear theem ntawm PCR tau yooj yim txheeb xyuas.Yog tias muaj ntau lub hom phiaj hom phiaj hauv Real-Time PCR, qhov pib yuav tsum tau teem rau txhua lub hom phiaj.Feem ntau, cov teeb liab fluorescence ntawm thawj 15 lub voj voog ntawm PCR cov tshuaj tiv thaiv yog siv los ua cov teeb liab fluorescence tom qab, thiab qhov pib fluorescence yog 10 npaug ntawm tus qauv sib txawv ntawm cov teeb liab fluorescence ntawm thawj 3 mus rau 15 lub voj voog ntawm PCR, thiab cov fluorescence pib yog teem rau hauv cov txheej txheem ntawm PCR.Feem ntau, txhua qhov ntsuas muaj nws qhov fluorescence pib ua ntej siv.
Cycle Threshold (CT) lossis Hla Point (CP)
Lub voj voog ntawm qhov kev nthuav dav nkhaus hla qhov chaw pib (piv txwv li, qhov taw tes ntawm qhov kev kuaj pom fluorescence nce ntxiv).CT tuaj yeem yog ib feem thiab tus nqi pib tus qauv tuaj yeem suav tau.Tus nqi CT sawv cev rau tus naj npawb ntawm cov voj voog uas tau ntsib thaum lub teeb liab fluorescent nyob rau hauv txhua lub PCR cov tshuaj tiv thaiv raj mus txog qhov chaw pib.Muaj kev sib raug zoo ntawm tus nqi CT ntawm txhua tus qauv thiab lub logarithm ntawm thawj tus qauv ntawm tus qauv,siab dua tus lej luam thawj, qhov me dua tus nqi CT, thiab rov ua dua.Ib qho qauv nkhaus tuaj yeem ua tau los ntawm kev siv tus qauv nrog tus lej luam thawj zaug, nyob rau hauv abscissa sawv cev rau CT tus nqi, thiab tus qauv sawv cev rau lub logarithm ntawm thawj tus lej luam.Yog li ntawd, ntev npaum li tus nqi CT ntawm tus qauv tsis paub yog tau txais, thawj tus lej luam ntawm tus qauv tuaj yeem suav los ntawm tus qauv nkhaus.
ΔCT tus nqi
ΔCT tus nqi piav qhiaqhov sib txawv ntawm lub hom phiaj gene thiab cov khoom siv endogenous siv cov noob CT tus nqi, xws li lub tsev tu noob, thiab yog siv los normalize tus nqi ntawm template siv:
⇒ΔCT = CT (lub hom phiaj gene) – CT (endogenous reference gene)
ΔΔCT Tus nqi
Tus nqi ΔΔCT piav qhia qhov sib txawv ntawm qhov nruab nrab ΔΔCT tus nqi ntawm cov qauv kev txaus siab (piv txwv li, cov hlwb txhawb nqa) thiab qhov nruab nrab ΔΔCT tus nqi ntawm cov qauv siv (xws li, cov hlwb tsis muaj zog).Cov qauv siv kuj tseem hu ua tus qauv calibration thiab tag nrho lwm cov qauv yog normalized rau qhov no rau qhov txheeb ze:
⇒ΔΔCT = nruab nrab ΔCT (piv txwv ntawm kev txaus siab) - nruab nrab ΔCT ( piv txwv)
Endogenous reference genes (cov noob caj noob ces)
Cov theem qhia ntawm endogenous reference genes, xws li tsev tu noob (tsev tu noob), tsis txawv ntawm cov qauv.Muab piv rau CT qhov tseem ceeb ntawm cov neeg siv cov noob rau lub hom phiaj gene tso cai rau theem qhia ntawm lub hom phiaj gene kom normalized rau tus nqi ntawm RNA lossis cDNA (saib ntu ntawm ΔCT qhov tseem ceeb saum toj no).
Internal reference genes raug raumuaj peev xwm RNA degradation los yog muaj cov enzyme inhibitors hauv RNA cov qauv, nrog rau kev hloov pauv hauv RNA cov ntsiab lus, rov qab ua kom zoo dua qub, nucleic acid rov qab, thiab cov qauv tuav.Txhawm rau xaiv qhov zoo tshaj plaws siv cov noob, peb tau hloov kho cov algorithm kom tso cai rau nws xaiv qhov kev siv zoo nyob ntawm qhov chaw sim.
Internal tswj
Ib qho kev tswj hwm uas tau nthuav dav hauv tib cov tshuaj tiv thaiv raws li lub hom phiaj ib ntus thiab soj ntsuam nrog ib qho kev sojntsuam sib txawv (piv txwv li, ua haujlwm duplex PCR).Kev tswj sab hauv feem ntau yog siv los txiav txim qhov ua tsis tau zoo, xws li thaum lub hom phiaj tsis raug kuaj pom.
Calibration Qauv
Ib qho qauv siv (piv txwv li, purified RNA los ntawm cov kab ntawm tes los yog cov ntaub so ntswg) siv nyob rau hauv cov txheeb ze kom muaj nuj nqis los sib piv tag nrho lwm cov qauv los txiav txim seb tus txheeb ze qhia theem ntawm ib tug noob.Cov qauv calibration tuaj yeem yog ib qho qauv, tab sis feem ntau yog kev tswj hwm (piv txwv li, tus qauv tsis kho lossis tus qauv ntawm lub sijhawm xoom ntawm qhov kev sim).
Kev tswj zoo
siv cov tshuaj tiv thaiv nrogpaub tus nqi ntawm template.Kev tswj zoo feem ntau yog siv los xyuas tias cov txheej txheem primer lossis primer-probe teeb ua haujlwm zoo thiab cov tshuaj tiv thaiv tau teeb tsa kom raug.
Tsis muaj Template Control (NTC)
Kev tswj cov tshuaj tiv thaiv uas muaj tag nrho cov khoom tsim nyog ntawm cov tshuaj amplification tsuas yog cov qauv, uas feem ntau yog hloov nrog dej.Kev siv NTC tuaj yeem pom cov kab mob tshwm sim los ntawm reagent contamination lossis txawv teb chaws DNA, yog li ua kom muaj tseeb thiab kev ntseeg siab ntawm cov ntaub ntawv tshawb pom.Amplification ntawm NTC tswj qhia tias muaj kab mob.
Tsis muaj RT tswj (NRT)
RNA extraction txheej txheem yuav muaj residual genomic DNA, uas yog heev teeb meem thiab yog tus culprit cuam tshuam cov ntaub ntawv zoo thiab lub ntuj yeeb ncuab ntawm qPCR, yog li thaum tsim thwmsim, nws yuav tsum tau tsim los tsuas amplify RNA nrhiav kom tau.Muaj ob txoj hauv kev, ib qho yog tsim cov primers thoob plaws introns, lwm qhov yog kom tshem tawm DNA kom tiav, qhov twg yog qhov zoo dua, uas yuav tau tham tom qab.NTR tswj yog daim iav khawv koob kom pom DNA muaj kuab lom.Yog hais tias muaj amplification, nws txhais tau hais tias muaj kuab paug.
Cov qauv
Cov qauv yog cov qauv ntawm kev paub txog concentration lossis tus lej luam uas siv los tsim cov qauv nkhaus.Txhawm rau kom muaj kev ruaj ntseg ntawm tus qauv, cov noob tawg feem ntau yog cloned rau hauv plasmid thiab siv raws li tus qauv.
Tus qauv nkhaus
feem ntau yog diluted rau hauv tsawg kawg yog 5 concentration gradients nrog cov qauv khoom raws li qhov sib piv ob npaug, thiab 5 cov ntsiab lus tau kos rau hauv kev sib koom ua ke ntawm CT tus nqi thiab tus lej luam, thiab cov ntsiab lus txuas nrog rau txoj kab los tsim cov qauv nkhaus.Rau txhua tus qauv nkhaus, nws qhov siv tau yuav tsum tau kuaj xyuas.Tus nqi nqes hav poob ntawm -3.3 thiab -3.8, thiab txhua qhov concentration yog ua nyob rau hauv triplicate.Cov ntsiab lus uas txawv ntawm lwm cov ntsiab lus yuav tsum muab pov tseg.Tus nqi CT ntawm tus qauv yuav tsum tau kuaj yog coj mus rau hauv tus qauv nkhaus, thiab cov theem qhia ntawm tus qauv yuav raug ntsuas tuaj yeem suav tau.
Tus nqi CT ntawm tus qauv yuav tsum tau kuaj yog coj mus rau hauv tus qauv nkhaus, thiab thawj tus lej luam ntawm tus qauv yuav raug ntsuas tuaj yeem suav tau.
Efficiency thiab nqes hav
Txoj kab nqes ntawm tus qauv nkhaus sawv cev rau kev ua haujlwm ntawm PCR lub sijhawm.
· Ib txoj kab nqes ntawm -3.322 qhia tias PCR amplification efficiency yog 1, los yog 100% npaum, thiab tus nqi ntawm PCR khoom ob npaug ntawm txhua lub voj voog.
· Ib txoj kab nqes qis dua -3.322 (piv txwv li, -3.8) qhia txog PCR efficiency
· Ib txoj kab nqes loj dua -3.322 (xws li, -3.0) qhia tias PCR efficiency zoo li ntau dua 100%, uas yog xav paub, yuav ua li cas ib lub voj voog ntawm PCR tsim tau ntau tshaj ob npaug ntawm cov khoom lag luam amplified?Qhov teeb meem no tshwm sim nyob rau hauv lub non-linear theem ntawm cov tshuaj tiv thaiv PCR, uas yog, muaj ib tug loj npaum li cas ntawm tsis-specific amplification.
melting nkhaus
Tom qab qPCR amplification tiav, cov khoom PCR yog rhuab.Raws li qhov kub thiab txias, cov khoom lag luam ob-stranded amplification maj mam yaj, ua rau txo qis hauv fluorescence siv.Thaum qhov kub thiab txias (Tm) mus txog, ntau cov khoom yuav yaj.Fluorescence poob sharply.Cov khoom PCR sib txawv muaj qhov sib txawv Tm qhov sib txawv thiab qhov sib txawv ntawm qhov kub thiab txias, yog li qhov tshwj xeeb ntawm PCR tuaj yeem txheeb xyuas.
Melting nkhaus (derivative curve)
Lub melting nkhaus yog muab los ua ib daim ntawv qhia ncov, uas muaj peev xwm ntau intuitively tso saib qhov teeb meem ntawm PCR cov khoom tawg.Txij li thaum lub melting kub yog Tm tus nqi ntawm DNA fragment, qee qhov tsis muaj feem cuam tshuam rau Tm tus nqi ntawm DNA fragment tuaj yeem txiav txim tau, xws li fragment loj, GC cov ntsiab lus, thiab lwm yam. Feem ntau hais lus, raws li peb cov qauv tsim primer,Qhov ntev ntawm cov khoom amplified yog nyob rau hauv thaj tsam ntawm 80-300bp, yog li lub melting kub yuav tsum nyob nruab nrab ntawm 80 ° C thiab 90 ° C.
Kev txhais ntawm lub melting nkhaus: Yog tias tsuas yog lub ncov tseem ceeb tshwm sim ntawm 80 ° C-90 ° C, nws txhais tau hais tias fluorescent quantitative PCR zoo meej;Yog hais tias lub ntsiab ncov tshwm ntawm 80 ° C-90 ° C thiab lwm yam ncov tshwm hauv qab 80 ° C, primer dimer yog qhov pib xav.Koj tuaj yeem sim nce qhov kub ntawm qhov annealing los daws nws;Yog tias lub ncov tseem ceeb tshwm nyob nruab nrab ntawm 80 ° C-90 ° C, thiab lwm yam ncov rov tshwm sim dua thaum kub nce, nws yog qhov pib xav tias muaj DNA paug, thiab DNA yuav tsum tau muab tshem tawm thaum pib ntawm qhov kev sim.
Tau kawg, tseem muaj qee qhov xwm txheej txawv txav, uas yuav raug rhuav tshem ib qho los ntawm ib qho hauv qab no.
3. Kev paub Advanced
Ua qPCR, kuv yuav tsum hais MIQE,Cov ntaub ntawv tsawg kawg nkausrau luam tawm ntawmMuaj nuj nqisReal-Time PCRKev sim - cov ntaub ntawv yam tsawg kawg nkaus rau kev tshaj tawm cov lus hais txog PCR lub sijhawm tiag tiagkev sim.Txhawm rau kom txhua tus neeg nkag siab yooj yim, peb yuav ua kom yooj yim cov ntsiab lus tseem ceeb.
Koj tuaj yeem tshawb nrhiav cov ntawv qub ntawm MIQE hauv Is Taws Nem, thiab qhov tseem ceeb tshaj plaws yog tias nws teev covcov ntaub ntawv txheeb xyuas uas yuav tsum tau muab thaum tshaj tawm ib tsab xov xwm .
Cov neeg soj ntsuam tuaj yeem txiav txim siab qhov zoo ntawm qhov kev sim los ntawm kev nyeem cov ntsiab lus no;Cov neeg nyeem yav tom ntej kuj tuaj yeem siv qhov no los rov ua dua lossis txhim kho qhov kev sim.
Nws yog tsim nyog sau cia tias hauv daim ntawv teev npe no, qhov tseem ceeb ntawm txhua daim ntawv teev npe yog cim nrog E lossis D.Nws txhais li cas?E: Cov ntaub ntawv tseem ceeb (yuav tsum xa tuaj);D: Cov ntaub ntawv xav tau (muab ntau li ntau tau).
MIQE (1) — Kev sim tsim
Ntau tus scumbags uas tau ua tiav lawv txoj kev tiv thaiv tom qab ua tiav lawv cov kev kawm tiav yuav tsis paub yuav ua li cas los tsim ib qho kev sim ntawm nws tus kheej, qhib lawv phau ntawv sau, thiab ua raws li tus kws qhia ntawv kom lawv ua.Raws li qhov tshwm sim, kev sim tsim tsis nruj, thiab lub tuam tsev kho mob ntawm cov ntawv xov xwm tau hais tias lawv xav ua daim duab no thiab daim duab ntawd, yog li lawv tau ua nws daze.Qhov no yog li cas cov scumbags tau ua!
Nyob ze rau tsev, thawj lub hauv paus ntsiab lus ntawm kev sim yog txiav txim siabqhov nruj ntawm kev sim logic.Qhov tseem ceeb tshaj plaws yog qhov kev sim tsim, thiab qhov tseem ceeb tshaj plaws ntawm kev sim tsim yog yuav ua li cas los teeb tsa lub hom phiaj, cov qauv siv (tswj), thiab tus lej ntawm kev rov ua dua, kom cov ntaub ntawv sim tuaj yeem hais txog, sib piv, thiab ntseeg tau.
Lub hom phiaj qauvyog hais txog cov qauv uas xav kom peb txheeb xyuas cov hom phiaj tom qab kev kho mob.Cov qauv sivyog cov qauv tsis muaj kev kho mob, uas feem ntau hu ua tsiaj qus hauv biology.
Kev sim replicatestseem ceeb heev.Feem ntau, tus naj npawb ntawm cov kev sib txuas lus yuav tsum muaj ntau tshaj peb.Nws yog ib qho tsim nyog yuav tsum paub qhov txawv ntawm dab tsi yog bioreplication thiab dab tsi yog technical replication.
Biological Replicates: Tib qhov kev sim ua pov thawj ua tiav nrog cov khoom sib txawv (lub sijhawm, cov nroj tsuag, cov khoom siv, cov tshuaj tiv thaiv daim hlau).
Biological duplication
Cia peb coj cov tshuaj tua kab ntawm cov kua txob ua piv txwv.Peb xav tshuaj tsuag tshuaj tua kab rau ntawm peb tsob nroj ntawm ABC, ces peb cov nroj tsuag ntawm ABC yog peb cov khoom siv roj ntsha, thiab lawv yog tib qho kev sim ua pov thawj ua nrog cov khoom sib txawv.Tab sis raws li kev sim, kev tswj hwm yog xav tau, yog li peb tuaj yeem txau ib qho ntawm cov ceg ntoo ntawm tsob ntoo A los ua ib pawg kev sim ntawm cov nroj tsuag A, thiab tsis yog tshuaj tsuag rau lwm ceg ntawm tsob ntoo A los ua pawg tswj hwm.Ua tib yam rau B thiab C.
Technical Replicates (Technical Replicates): Nws yog ib qho kev sim rov qab tsim los kom tsis txhob muaj qhov yuam kev los ntawm kev ua haujlwm, uas yog qhov sib npaug ntawm cov khoom siv tib yam.Ob qho kev kho mob thiab kev tswj hwm yuav tsum muaj qhov chaw rov ua dua (yam tsawg kawg peb) ntawm lub hom phiaj cov noob thiab cov noob siv sab hauv.
Kev rov ua dua tshiab
Siv cov kua txob kho nrog tshuaj tua kab ua piv txwv dua.Rau cov kev sim ntawm cov nroj tsuag A, peb tau ua peb lub PCR qhov ntawm 1, 2, thiab 3 rau nws lub hom phiaj gene thiab sab hauv siv cov noob raws li qhov nruab nrab tom qab kuaj pom.Rau kev tswj ntawm cov nroj tsuag A Pawg kuj raug kho tib yam.Ib yam li ntawd, ua tib yam kev kho mob rau cov nroj tsuag B thiab C.Qhov no yog technical repetition.
Nws tsim nyog sau cia tiasdab tsi nkag mus rau hauv cov txheeb cais yog cov khoom siv roj ntsha rov ua dua, thiab cov kev rov ua dua tshiab yog los sim seb puas muaj cov xwm txheej tshwm sim hauv cov txheej txheem sim, txhawm rau ua kom cov txiaj ntsig kev sim tau lees paub, uas yog, kom tsis txhob ua yuam kev los ntawm kev noj lawv qhov nruab nrab raws li peb feem ntau hais.
Kev tswj tsis zoo - NTC thiab NRT
NTC (No-Template Control), ib qho kev tswj tsis muaj tus qauv, yog siv los xyuas seb cov khoom siv sim puas muaj paug.Feem ntau, dej yog siv los ua tus qauv.Yog tias muaj cov tshuaj tiv thaiv fluorescent, nws qhia tau tias muaj kab mob nucleic acid tshwm sim hauv chav kuaj.
Cov pa phem no los ntawm: dej tsis huv, tsis tsim nyog reagents uas muaj endogenous DNA, primer pollution, kuaj cov khoom pollution, aerosol pollution, thiab lwm yam, yuav tsum tau siv RNase scavengers thiab RNase inhibitors.Aerosol muaj kuab paug yog qhov nyuaj nrhiav tau.Xav txog tias koj lub chaw kuaj mob zoo li smog, nrog ntau yam nucleic acids raug tshem tawm hauv huab cua.
NRT (No-Reverse Transscriptase), kev tswj tsis tau rov qab hloov dua siab tshiab, yog qhov tsis thim rov qab RNA raws li kev tswj tsis zoo, uas yog kev tswj hwm ntawm gDNA residue.
Thaum ua cov kev qhia gene, tus nqi ntawm RNA raug kuaj pom los ntawm kev kuaj xyuas tus nqi ntawm cDNA tom qab kev hloov pauv rov qab.Yog tias muaj gDNA residue thaum RNA raug ntxuav, nws yuav ua rau muaj qhov yuam kev hauv qhov kev sim, vim tias qhov tseeb tau txais yog gDNA thiab cDNA.Nyob rau theem sib sau, tsis yog cDNA nkaus xwb, gDNA yuav tsum tau muab tshem tawm tag nrho thaum RNA rho tawm.
MIQE (2)—cov ntaub ntawv piv txwv
Cov ntaub ntawv hu ua piv txwv txhais tau hais tias thaum peb tshaj tawm ib tsab xov xwm txog qPCR, peb yuav tsum piav qhia cov qauv ntaub ntawv kom meej, uas yog ib feem tseem ceeb ntawm tsab xov xwm.Ib yam li ntawd, thaum peb ua cov qauv, peb kuj yuav tsum tswj peb tus kheej kev ua haujlwm kom ntseeg tau tias qhov siv tau ntawm cov qauv.
Cov lus piav qhia ntawm tus qauv tsuas yog qhov tshwm sim, thiab peb yuav tsum tau them sai sai rau cov khoom siv thaum lub sijhawm sim tag nrho.
Xaiv cov khoom siv sim
Cov ntshav kuaj - xaiv cov ntshav tshiab, tsis pub dhau 4 teev.Cell kuaj - xaiv los sau cov hlwb tshiab nyob rau lub sijhawm ntawm kev loj hlob zoo.Tsiaj Tissue - Xaiv cov ntaub so ntswg tshiab, nquag loj hlob.Cog Tissue - Xaiv cov ntaub so ntswg tshiab, hluas.
Koj yuav tsum tau pom tias muaj ib lo lus tseem ceeb hauv ob peb kab lus no: tshiab .
Rau cov qauv saum toj no, qhov zoo tshaj plaws, muaj txiaj ntsig zoo, thiab cov khoom siv ruaj khov ntawm kev ua lag luam yog Foregene cov khoom siv, uas tuaj yeem tshem tawm lawv cov DNA thiab RNA sai thiab yooj yim.
Tsiaj Tag Nrho RNA Isolation Kit
Plant Total RNA Isolation Kit Plus
Kev khaws cia cov ntaub ntawv sim
Feem ntau hais lus, peb tsis pom zoo kom khaws cov qauv, yog tias muaj kev tso cai.Txawm li cas los xij, muaj ntau tus phooj ywg uas tsis tuaj yeem ua qhov kev sim tam sim tom qab kuaj, thiab qee qhov tseem yuav tsum nqa cov kua nitrogen tso tsheb hlau luam mus rau qhov chaw ua piv txwv.
Rau cov phooj ywg zoo li no, kuv tsuas hais tau tias koj tsis nkag siab txog cov khoom siv reagent.Tam sim no ntau reagent consumable tuam txhab uas muag tsim reagents uas tuaj yeem khaws cov qauv RNA ntawm chav sov, thiab koj tuaj yeem xaiv siv lawv.Txoj kev khaws cia yog cov kua nitrogen cia, siv cov kua nitrogen me me uas yooj yim nqa.Tom qab nqa cov qauv rov qab mus rau qhov chaw kuaj, muab tso rau hauv lub tub yees -80 ° C.
Rau kev sim nrog RNA, rau lo lus hauv paus ntsiab lus yuav tsum ua raws li:tsis kub, tsis muaj enzymes,thiabceev .
Lub tswv yim ntawm qhov kub thiab txias yog yooj yim to taub;tsis muaj enzymes, RNase yog nyob txhua qhov chaw hauv ntiaj teb peb nyob hauv (tsis li ntawd koj yuav raug tua los ntawm HIV), yog li yuav ua li cas kom tsis txhob RNase thaum ua kev sim yog lub tswv yim tseem ceeb heev;ceev ceev,Tsis muaj Kung Fu hauv ntiaj teb uas tsis tuaj yeem tawg, tsuas yog ceev tsis tuaj yeem tawg.
Yog li ntawd, nyob rau hauv ib qho kev txiav txim, lub sij hawm luv luv lub extraction, qhov zoo dua cov khoom siv.Vim li casForegene's cov khoom siv qhia ceev ceev, vim lawv paub zoo.
PS: Qee tus ntxhais ua qhov kev sim ua tib zoo, tab sis lawv tsis zoo li lub slam dunk tom qab ntau xyoo ua haujlwm.Lawv xav tias Vajtswv tsis ncaj ncees, yws txog lwm tus, thiab nrhiav txoj sia.Qhov tseeb, nws tsis to taub.Nws tsis tiv thaiv RNA zoo, thiab tus neeg ua si slam dunk tau nrawm.Thaum nws tab tom ua qhov kev sim, nws xav tias nws yuav ua tiav qhov slam dunk nrog peb zaug, tsib zaug thiab ob ntu, tab sis nws ua qhov kev sim ua kom tiav.
Nco tseg: Tsawg dua, muaj feem ntau ntawm RNase ntxeem tau.Yuav ua li cas cob qhia koj tus kheej kom ceev?Tsis muaj txoj hauv kev, tsuas yog xyaum ntxiv.
Rau cov kev sim sib txawv thiab cov qauv sib txawv, nws tseem yuav tsum tau nyeem ntau cov ntaub ntawv thiab xaiv ib txoj hauv kev tsim nyog rau kev ua.Rau cov qauv sau thiab khaws cov txheej txheem, MIQE xav kom nws yuav tsum tau sau kom meej hauv daim ntawv, kom cov neeg soj ntsuam tuaj yeem tshuaj xyuas qhov kev ntseeg siab ntawm daim ntawv, thiab nws kuj yog qhov yooj yim rau cov menyuam yaus uas xav tsis thoob los rov ua koj qhov kev sim.
Txawm hais tias kev sim tshuaj lom neeg yog qhov nyuaj, lawv yog high-end.Yog tias koj tsis ceev faj, koj tuaj yeem hla lub ntiaj teb.Piv txwv li, ua rau SARS mus rau hauv cov teeb meem biochemical, lossis ua hybrid nplej kom txuag tau 1.3 billion tus neeg.Daim duab hauv qab no yog kev sim tshuaj lom neeg, koj yuav tsum nkag siab tias koj txaus siab npaum li cas ntawm koj qhov kev tshawb fawb tsuas yog saib nws zoo li dick.Tsis nco qab nws, tsis txhob dub nws.
MIQE (3) - nucleic acid extraction.
Nucleic acid extraction yog ib qho kev tshwm sim loj, thiab txhua qhov kev sim molecular biology pib nrog nucleic acid extraction.Ua ntej tshaj plaws, cia peb luam MIQE cov ntsiab lus ntawm nucleic acid extraction.
Saib ntawm daim ntawv no, koj tsis tuaj yeem nyob ntawm qhov chaw.Daim ntawv yog dogma.Txhawm rau ua ib tus tub ntxhais kawm sab saum toj, koj yuav tsum nug yog vim li cas.Cov ntsiab lus tseem ceeb ntawm cov lus no yog: Ua rawsLub purity, kev ncaj ncees, sib xws, thiab rho tawm tus nqi ntawm RNA .
Thawj ntu ntawm covtxheej txheem los yog ntsuas yog lub nucleic acid extraction kauj ruam.Yog tias koj siv lub tshuab tsis siv neeg nucleic acid extractor (advanced, thov hu rau kuv rau kev yuav khoom), koj yuav tsum qhia tus qauv lub npe ntawm qhov ntsuas.
Lub npe ntawm cov khoom siv thiab
cov khoom siv dab tsi tau siv rau cov ntsiab lus hloov pauv, dab tsi tshwj xeeb reagents tau ntxiv lossis ua haujlwm tshwj xeeb dab tsi yuav tsum tau piav qhia kom meej kom lwm tus tuaj yeem yooj yim rov ua dua koj qhov kev sim.
Qee tus neeg ntxiv qee qhov tshwj xeeb reagents thaum rho cov qauv tshwj xeeb, xav tias qhov no yog lawv cov riam phom zais cia thiab tsis txhob qhia rau lwm tus.Thaum khaws cia tsis pub leejtwg paub, lawv kuj poob lub sijhawm los ua kom koj tsab xov xwm ci ntsa iab.Tsis txhob ntse, koj yuav tsum ua siab ncaj tshaj lub teb chaws qub Zhang nyob rau hauv kev tshawb fawb, yog hais tias koj xav kom ntse, tsab xov xwm yuav ua rau koj ruam.
yuav tsum nco ntsoov cov khoom naj npawb ntawm cov khoom sivthaum koj xaj cov khoom siv thiab sau cov ntawv.Feem ntau muaj ob tus lej ntawm cov khoom siv: Cat-catalog naj npawb (cov khoom naj npawb, tsab xov xwm), Lot-cov khoom ntau tus lej (Siv los qhia seb cov khoom twg tuaj ntawm).
Tsis tas li ntawd, tus lej CAS feem ntau siv thaum xaj cov tshuaj biochemical reagents, thiab kuv yuav nrov nws ua ke.Tus lej CAS yog tus lej muab los ntawm American Chemical Society rau txhua yam tshuaj tshiab.Feem ntau, peb tus lej txuas nrog tus dash.CAS Nr .: 7732-18-5Cov tshuaj feem ntau muaj ntau lub npe, tab sis CAS tus lej yog qhov tshwj xeeb.Thaum xaj tshuaj, koj tuaj yeem tshawb xyuas nws tus lej CAS ua ntej.
Nyob ze rau tsev, ua cas peb yuav tsum piav tej no kom meej?Qhov tseeb, nws kuj yog los xyuas qhov zoo ntawm RNA extraction.Kev siv cov cuab yeej thiab cov khoom siv yuav ua rau RNA rho tawm zoo ib yam.Cov txheej txheem rho tawm ntawm cov chaw sim zoo tib yam tsis loj, thiab tuaj yeem tau txais nrog cov khoom siv.
Cov ntsiab lus ntawm kev kho DNase lossis RNase
Qhov teeb meem tseem ceeb ntawm fluorescent quantitative PCR yog tiv thaiv DNA paug, thiab tsis txhob sim yog tias muaj kab mob.Yog li ntawd, nws yog ib qho tsim nyog los hais txog cov txheej txheem uas koj tau siv los ua cov DNA, txhawm rau ua kom pom tias DNA hauv cov txheej txheem sim tau raug tshem tawm tag nrho.sawv cev los ntawm daim duab schematic.
Schematic daim duab ntawm RNA thiab DNA
Feem ntau, txoj kev tshem tawm DNA yog kho RNA nrog DNase tom qab rho tawm.Txawm li cas los xij, cov no yog cov txheej txheem qub.Kev lag luam RNA cov khoom siv rho tawm tau tuaj yeem tshem tawm DNA thaum lub sijhawm rho tawm yam tsis tau ntxiv DNase.Piv txwv li, cov khoom siv los ntawm Foregene .
Nco tseg: Tshem tawm DNA thaum RNA rho tawm yog ib qho phom sij txaus ntshai heev, uas yuav ua rau lub sijhawm ua haujlwm ntawm RNA rho tawm thiab ua rau muaj kev pheej hmoo ntawm RNA degradation.Yeej, nws yog kev lag luam tawm ntawm RNA tawm los thiab purity.
Tsis tas li ntawd, tus nqi ntawm DNase ntxiv rau silica-raws li adsorption kem yog tsawg heev, thiab zoo DNase yuav tsum tau siv los ua kom tiav cov nyhuv.Unoptimized DNase tsis tuaj yeem zom tau sai thiab tag.Qhov no yog qhov kev ntsuam xyuas ntawm cov tub lag luam cov txheej txheem.Tau kawg, muaj ntau tus tub lag luam uas khav theeb tias DNA tuaj yeem raug tshem tawm yam tsis muaj DNase.Nws tuaj yeem hais tias leej twg brags tias DNA tuaj yeem raug tshem tawm tag nrho yam tsis muaj DNase yog hooligan.DNA yog ib qho ruaj khov ob-stranded qauv, thiab nws tsis tuaj yeem raug tshem tawm los ntawm kev hais lus thiab luag.
Kev ntsuam xyuas kev kis kab mob
Kev soj ntsuam txoj kev: electrophoresis nrhiav pom, 1% agarose, 6V / cm, 15min, loading 1-3 ul
Nucleic acid quantitative tsom xam
Feem ntau yog ntsuas siv UV spectrophotometer.Cia kuv ua ntej nrov lub ntsiab lus ntawm peb qhov tseem ceeb ntawm OD260, OD280, thiab OD230.
· OD260nm: Nws yog qhov nqus dej ntawm qhov siab tshaj plaws nqus ntawm nucleic acid, thiab qhov ntsuas qhov zoo tshaj plaws ntawm 0.1 txog 1.0.Yog tias tsis yog, dilute lossis mloog zoo rau cov qauv coj mus rau hauv qhov ntau.
· OD280nm: Nws yog qhov nqus dej ntawm lub siab tshaj plaws nqus cov protein thiab cov tshuaj phenolic.
· OD230nm: Nws yog qhov nqus dej ntawm lub siab tshaj plaws nqus ntawm carbohydrates.
Tom ntej no, cia peb tham txog lub luag haujlwm ntawm txhua qhov ntsuas.Rau A260, nws tuaj yeem siv los ntsuas cov txiaj ntsig ntawm nucleic acid.Thaum OD260 = 1, dsDNA = 50μg / ml, ssDNA = 37μg / ml, RNA = 40μg / ml.
Rau purity, peb yuav tsum tau saib cov piv txwv uas peb nquag pom: OD260/280 thiab OD260/230.
· Pure DNA: OD260/280 yog kwv yees li ntawm 1.8.Thaum nws loj dua 1.9, nws qhia tau hais tias muaj RNA muaj kuab paug, thiab thaum nws tsawg dua 1.6, nws qhia tau hais tias muaj protein ntau thiab phenol muaj kuab paug.
· Ntshiab RNA: 1.7
· OD260/230: Txawm nws yog DNA lossis RNA, tus nqi siv yog 2.5.Thaum nws tsawg dua 2.0, nws qhia tau tias muaj kuab paug ntawm qab zib, ntsev thiab organic teeb meem.
RNA kev ncaj ncees
Nws yog ib qho tseem ceeb heev los ntsuas qhov ncaj ncees ntawm RNA.Feem ntau, nws yog ib qho tsim nyog yuav tau ua qhov kev sim RNA denaturation gel los xyuas seb qhov ci ntawm 28S thiab 18S RNA yog ob txoj kev sib raug zoo.Thaum lub thib peb band 5S tshwm, nws txhais tau hais tias RNA tau pib degrade, tshwj tsis yog rau invertebrates.
Cov ntaub ntawv rau RNA kev ntsuam xyuas zoo: Ntxiv rau cov kev ntsuam xyuas saum toj no, kuj tseem muaj qee qhov kev ntsuas ntsuas siab dua ntawm RNA kev ncaj ncees, xws li RQI kev ntseeg siab ntawm Experion tsis siv neeg electrophoresis system, uas tuaj yeem kuaj xyuas seb RNA puas yog degraded invisibly.
Hauv kev tshawb fawb tshawb fawb, fluorescent quantitative PCR yog kev sib piv ntawm lub hom phiaj gene thiab cov gene hauv cov ntaub ntawv.Yog li ntawd, nyob rau hauv tus txheej txheem ntawm RNA qauv preservation, RNA extraction, thiab lwm yam., lub hom phiaj tseem ceeb yog los xyuas kom meej qhov kev ncaj ncees ntawm RNA.
Yuav ua li cas qhov kev ncaj ncees ntawm RNA cuam tshuam qhov sib npaug ntawm lub hom phiaj gene thiab lub hauv paus siv cov noob tuaj yeem nkag siab yooj yim los ntawm daim duab hauv qab no.Kev degradation yuav ua rau cov noob tsis tiav, txawm tias nws yog qhov ua tsis tiav ntawm cov khoom siv hauv cov noob los yog qhov tsis tiav ntawm cov hom phiaj noob, nws yuav muaj kev cuam tshuam zoo rau cov ntaub ntawv.
Schematic daim duab ntawm lub hom phiaj gene thiab siv cov noob, yuav tsum tsis muaj tseeb
Inhibition test (txawm tias tus nqi CT raug txwv nyob rau hauv siab lossis qis concentration lossis lwm yam mob)
Siv daim duab no ua piv txwv, Ct qhov tseem ceeb ntawm tsib nkhaus yog raws li hauv qab no.Kev faib tawm ntawm CT qhov tseem ceeb ntawm qhov nkhaus yog qhov tsis sib xws, thiab Ct qhov tseem ceeb tau ncua sijhawm nyob rau hauv qhov siab thiab qis qis, uas yog cov ntaub ntawv ntawm PCR inhibition.
Lub ntsiab lus tseem ceeb: Hauv cov txheej txheem ntawm RNA rho tawm, peb yuav tsum tso tseg qhov kev xav tsis zoo thiab tsim kom muaj qhov tseeb.
Lub tswv yim tsis ncaj ncees yog: RNA rho tawm tsuas yog ua raws cov txiaj ntsig, xav tias qhov loj ntawm RNA tau txais, qhov zoo dua.Qhov tseeb, thaum peb ua kom muaj nuj nqis, yog tias tus lej ntawm cov noob tsis loj heev, peb tsis xav tau RNA ntau.Tus nqi ntawm RNA koj rho tawm yog ntau tshaj li txaus.
Lub tswvyim tiag tiag yog:Kev rho tawm RNA yuav tsum nrhiav kev purity, kev ncaj ncees thiab sib xws.Purity tuaj yeem ua kom ntseeg tau tias cov ntaub ntawv rov qab rov qab tsis raug cuam tshuam thiab cov ntaub ntawv yuav tsis cuam tshuam los ntawm DNA.Kev ncaj ncees ua kom muaj kev sib npaug ntawm cov phiaj xwm sib luag thiab cov ntaub ntawv sab hauv.Consistency kom ruaj khov qauv loading.
MIQE (4) - thim rov qab
Kev xav yuam kev: nrhiav cov qauv ntim ntau dua.
Yog tswvyim: Ua raws li qhov sib xws (kev ruaj ntseg), tsis hais tus nqi ntawm RNA loaded, qhov ua tau zoo ntawm kev hloov pauv rov qab tseem zoo ib yam, kom ntseeg tau tias qhov sib txawv hauv cDNA tuaj yeem cuam tshuam qhov sib txawv ntawm mRNA.
Peb piav qhia txog txheej txheem no nrog daim duab schematic:
Schematic daim duab ntawm rov qab transcription efficiency, tsis muaj tseeb
Ua ntej tshaj plaws, peb yuav tsum nkag siab qhov sib txawv ntawm cov txheej txheem thim rov qab thiab txheej txheem PCR.PCR tau dhau los ua ntau yam cua sov thiab cov txheej txheem annealing, thiab lub hom phiaj fragment hlob exponentially;thaum thim rov qab tsis muaj cov txheej txheem no, peb tuaj yeem xav tias qhov kev hloov pauv rov qab yog qhov tseeb ib-rau-ib Lub Sijhawm rov ua dua, raws li ntau daim ntawm RNA
Raws li muaj peev xwm tau txais ntau daim ntawm cDNA Cov Ntaub Ntawv, nws yuav tsum nkag siab tam sim no, vim tias cov fragments loj thiab me tau thim rov qab, thiab nws tsis tuaj yeem tsom mus rau ib feem.Thiab vim hais tias tus nqi ntawm RNA yog me me, tus nqi ntawm cDNA tau txais kuj tseem me me, tsis zoo li PCR, uas muaj cov nyhuv amplification, yog li nws tsis yooj yim sua kom kuaj tau.
Cov txiaj ntsig ntawm cDNA electrophoresis
Qhov thib ob, qhov zoo tshaj plaws, thim rov qab transcription yog ua ib-rau-ib, tab sis tsis muaj rov qab transcriptase los ntawm ib lub tuam txhab tuaj yeem ua tiav cov txiaj ntsig no.Yeej, qhov ua tau zoo ntawm feem ntau thim rov qab transcriptases wanders ntawm 30-50%.Yog hais tias qhov no yog qhov teeb meem, peb xav kom muaj qhov ruaj khov rov qab ua haujlwm tau zoo, uas yog qhov peb xav pom hauv daim duab: 3 RNAs tau 2 cDNAs, 6 RNAs tau 4 cDNAs, yog li tsis muaj teeb meem pes tsawg tus qauv yog loaded, qhov rov qab transcription efficiency kuj ruaj khov.Peb tsis xav pom qhov xwm txheej uas qhov kev hloov pauv rov qab ua haujlwm tsis ruaj khov thiab siab concentration yog inhibited.
Yog li, yuav ua li cas los txheeb xyuas seb qhov kev hloov pauv hloov pauv tau zoo li cas?Cov txheej txheem yog yooj yim heev, koj tsuas yog yuav tsum tau ua qhov kev sib piv: ib qho yog rov qab sau rau hauv cDNA tom qab ob npaug dilution ntawm RNA, thiab lwm yam yog ua kom ob npaug dilution tom qab thim rov qab mus rau cDNA, thiab tom qab ntawd ua qPCR kom pom qhov tau txais nqes hav puas zoo ib yam.Raws li cov tub ntxhais kawm sab saum toj, koj yuav tsum nkag siab nws hauv vib nas this.Raws li qhia hauv qab no:
Dilution ntawm RNA thiab cDNA los ntsuam xyuas seb qhov ua tau zoo ntawm kev hloov pauv rov qab yog qhov ruaj khov
Rov qab transcriptase thiab cov khoom siv
Yuav ua li cas thiaj li zoo meej fluorescent quantitative PCR muaj qhov zoo tshaj plaws rov qab transcriptase thiab cov khoom siv.Rov qab transcriptase yog kwv yees muab faib ua ob hom raws li qhov chaw, AMV lossisM-MLV, thiab lawv qhov kev ua tau zoo yog tib yam li cov lus qhia.
RNase H ua haujlwm
RNase H yog Ribonuclease H, Suav lub npe yog ribonuclease H, uas yog endoribonuclease uas muaj peev xwm tshwj xeeb hydrolyze RNA hauv DNA-RNA hybrid saw.RNase H tsis tuaj yeem hydrolyze cov phosphodiester bonds nyob rau hauv ib leeg-stranded los yog ob-stranded DNA los yog RNA, uas yog, nws yuav zom tsis tau ib leeg-stranded los yog ob-stranded DNA los yog RNA.Feem ntau siv rau hauv kev sib txuas ntawm ob txoj hlua ntawm cDNA.
Nws yog ib qho txawv.Peb hais tias thim rov qab transcriptase muaj RNase H kev ua, tsis yog qhov rov qab transcriptase muaj RNase H, thiab nws yuav tsis tuaj yeem cais RNase H los ntawm thim rov qab transcriptase, tej zaum vim yog kev ua raws ntawm qee pawg hauv kev thim rov qab transcriptase Qhov kev ua no yog tshwm sim los ntawm kev rov qab transcriptase.
Yog li ntawd, tsis hais qhov kev hloov pauv ntau dua ntawm AMV, nws cov haujlwm RNase H txo cov txiaj ntsig ntawm cDNA.Tau kawg, reagent manufacturers yog niaj hnub optimizing lawv cov khoom kom tshem tawm RNase H kev ua nyob rau hauv thim rov qab transcriptase ntau npaum li ua tau kom cov yield ntawm cDNA.
Annealing kub
Cov qauv thib ob ntawm RNA ntawm qhov kub sib txawv
Saib daim duab saum toj no rau cov qauv thib ob ntawm RNA ntawm qhov sib txawv ntawm qhov kub thiab txias, thiab siv mFold online cuab yeej los txiav txim siab tus qauv thib ob ntawm lub hom phiaj tawg nyob rau hauv qhov tshwj xeeb kub thiab ntsev concentration.Ntawm 55 ° C, cov qauv thib ob ntawm RNA tseem yog qhov nyuaj heev, rov qab transcriptase tsis tuaj yeem ua haujlwm, thiab cov qauv thib ob tsis tuaj yeem daws tau tag nrho txog 65 ° C, thaum qhov zoo tshaj plaws kub ntawm AMV thiab M-MLV yog qis dua qhov ntsuas kub no.
yuav ua li cas?Cov qauv thib ob yog kev sib koom ua ke ntawm tus qauv nws tus kheej, uas ua rau muaj kev sib tw muaj zog ntawm cov primer thiab thim rov qab transcriptase thiab cov qauv, uas ua rau muaj teeb meem xws li qis E thiab tsis zoo rov ua dua.
yuav ua li cas?Tsuas yog nce qhov kub ntawm annealing kom ntau li ntau tau.
Ntau lub tuam txhab reagent tab tom txhim kho lawv qhov rov qab transcriptase los ntawm genetic engineering.Qee qhov ua rau muaj qhov kub thiab txias, xws li Jifan thiab Aidelai, thiab qee qhov tshem tawm cov pab pawg nquag ntawm RNase H enzyme los txhim kho kev sib raug zoo ntawm enzyme thiab RNA template.High affinity tuaj yeem sib tw nyem tawm cov qauv thib ob thiab nyeem tau yooj yim, thiab tseem txhim kho qhov ua tau zoo ntawm kev thim rov qab.
Cov ntsiab lus tseem ceeb: Rov qab cov ntaub ntawv rov qab yog qhov tseem ceeb dua kom ua raws li qhov sib xws ntawm kev rov qab ua kom zoo dua qub (enzymes yuav tsum tsis yog tsuas yog ua tau zoo tab sis kuj ruaj khov), ntau dua li tus nqi ntawm cov qauv thauj khoom, yog tias nws tsis yog qhov tshwj xeeb loj fluorescent quantitative PCR, nws yuav ua tsis tau txhua.Ntau cDNAs.
Ntau lub tuam txhab kuj tau ua qee qhov kev siv zog hauv kev nrhiav kev sib xws.Piv txwv li, feem ntau cov tuam txhab tam sim no tau ntim cov ntawv rov qab los ua cov khoom siv rau kev muag khoom, uas yog qhov kev xaiv zoo.
Piv txwv li, Foregene's RT Easy Series cov khoom siv:
RT Easy I (Master premix rau thawj strand cDNA synthesis kit)
MIQE (5) - hom phiaj cov ntaub ntawv
Daim duab saum toj no piav qhia
1. Txawm hais tias cov noob no muaj txiaj ntsig zoo rau kev sim rov ua dua, feem ntau tuaj yeem txheeb xyuas tau los ntawm kev sim dua.
2. Gene ID, koj paub.
3. Gene ntev, tag nrho ntev ntawm lub hom phiaj noob yog twv yuav raug hu tsis muaj teeb meem.Thaum tsim cov primers, xyuas kom meej tias qhov ntev ntawm lub amplicon yog nyob nruab nrab ntawm 80-200bp los xyuas kom meej qhov zoo ntawm amplification efficiency.
4. Kev sib piv cov ntaub ntawv sib piv, lub hom phiaj ntawm cov noob yuav tsum tau muab piv rau hauv genebank kom tsis txhob muaj qhov tshwj xeeb amplification.
5. Muaj pseudogenes.Lub pseudogene yog DNA ib ntus zoo ib yam li cov noob ib txwm tab sis poob nws txoj haujlwm ib txwm muaj.Nws feem ntau muaj nyob hauv ntau tsev neeg ntawm eukaryotes.Nws feem ntau yog sawv cev los ntawm ψ.Nws yog ib qho tsis ua haujlwm genomic DNA luam hauv genome uas zoo ib yam li coding gene sequence., feem ntau tsis sau, thiab tsis muaj lub ntsiab lus meej ntawm lub cev.
6. Txoj hauj lwm ntawm primers txheeb ze rau exons thiab introns.Nyob rau hauv thaum ntxov xyoo, thaum peb daws tau qhov teeb meem ntawm DNA paug, peb feem ntau them nyiaj mloog mus rau txoj hauj lwm ntawm primers, exons, thiab introns, thiab feem ntau txiav txim siab tsim primers nyob rau hauv introns kom tsis txhob DNA amplification.Thov saib daim duab hauv qab no: dub sawv cev rau introns, ntau yam blues sawv cev exons, liab dawb sawv cev rau cov primers, thiab ci liab sawv cev rau intron-spanning primers.
Schematic, yeej tsis muaj tseeb
Lub tswv yim zoo li cas no zoo li, tab sis qhov tseeb, feem ntau, cov trans-intron primers tsis zoo li tej yam yees siv, thiab lawv kuj yuav ua rau tsis muaj kev nthuav dav.Yog li txoj hauv kev zoo tshaj plaws los tiv thaiv DNA kab mob yog tshem tawm DNA tag.
7. Kev ua raws li kev twv ua ntej.Siv qhov piv txwv no dua, siv mFold online cuab yeej los txiav txim siab tus qauv thib ob ntawm lub hom phiaj tawg ntawm qhov tshwj xeeb kub thiab ntsev concentration.
Cov qauv thib ob ntawm RNA ntawm qhov kub sib txawv
Cov txheej txheem thib ob yog kev sib koom ua ke ntawm tus qauv nws tus kheej, uas yuav ua rau muaj kev sib tw ntawm cov primer thiab template pairing, thiab cov kev pheej hmoo ntawm primer binding tsawg dua, uas ua rau muaj teeb meem xws li qis E thiab tsis zoo repeatability.Los ntawm kev twv ua ntej software, yog tias tsis muaj qhov teeb meem thib ob, qhov ntawd yuav zoo heev.Yog tias muaj, peb tsab xov xwm txuas ntxiv yuav tshwj xeeb tham txog yuav daws qhov teeb meem no li cas.
MIQE (6)-qPCR Oligonucleotides
Rau fluorescent quantitative PCR, thawj qhov uas koj tawm tsam nrog txhua hnub yog RNA rho tawm, thiab qhov thib ob yuav yog tus qauv tsim.
Ua ntej tshaj plaws, peb tseem tshawb xyuas cov cai ntawm kev tsim primer raws li daim ntawv teev npe MIQE.Nws yog qhov yooj yim heev uas cov scumbags tuaj yeem luag, thiab peb tuaj yeem ua tiav hauv ib kab lus: nrhiav qhov sib lawv liag thiab txoj hauj lwm ntawm primer sojntsuam thiab cov kev hloov kho.Rau cov txheej txheem purification primer, primer synthesis yog li pheej yig tam sim no, qPCR tsim nyog ntawm PAGE thiab saum toj no purification txoj kev, thiab cov ntaub ntawv ntawm lub synthesis instrument tsis tseem ceeb.Ntau tus neeg tau ua primers rau ntau xyoo lawm thiab tsis paub tias cov khoom siv hluavtaws yog ABI3900.
Hais txog cov hauv paus ntsiab lus ntawm primer tsim, koj tsis tas yuav cim lawv los ntawm rote, vim hais tias feem ntau primer tsim software lossis cov cuab yeej hauv online tuaj yeem saib xyuas cov teeb meem no (pom zoo hauv online tool primer3.ut.ee/), thiab 99.999% ntawm primer design tsis ua manually Saib, tus sau qee zaum tsim ntau pua primers ib hnub, yog tias koj nyeem ib los ntawm ib qho, nws yuav dhau mus.
Tsuas yog xyuas cov ntsiab lus hauv qab no tom qab cov primers tsim:
1. Tsim primers ze rau 3′ kawg: Nyob rau hauv cov ntaub ntawv ntawm kev siv oligo dT primers rau cDNA thawj-strand synthesis, xav txog qhov rov qab transcription efficiency thiab RNA kev ncaj ncees, tus tsim primers yuav tsum tau tsim nyob ze rau 3′ kawg los txhim kho amplification efficiency.Siv daim duab los piav raws li hauv qab no (tsis muaj txoj hauv kev los nkag siab qhov no):
Vim li cas cov primers yuav tsum tau tsim nyob ze rau 3′ kawg, nws yuav tsum tsis muaj tseeb
2. TM tus nqi: Tus nqi Tm yog nyob ntawm 55-65 ° C (vim cov kev ua ntawm exonuclease yog siab tshaj ntawm 60 ° C), thiab cov ntsiab lus GC yog 40% -60%.
3. BLAST: Txhawm rau kom tsis txhob muaj kev nthuav dav ntawm cov genome, Blast yuav tsum tau siv rau kev txheeb xyuas ntxiv.
MIQE(7)-qPCR txheej txheem
1. qPCR cov khoom siv
Raws li qhov yuav tsum tau ua ntawm MIQE, peb yuav tsum tau piav qhia meej txog cov kev ua tiav cov tshuaj tiv thaiv hauv tsab xov xwm, suav nrog kev teeb tsa ntawm PCR cov tshuaj tiv thaiv kab mob, cov khoom siv dab tsi yog siv, leej twg yog tus tsim khoom, qhov loj npaum li cas ntawm cov tshuaj tiv thaiv kab mob, txawm tias txoj kev zas xim los yog txoj kev sojntsuam yog siv, PCR program settings.Cov neeg tsav tsheb qub yuav pom tau tias ntev npaum li cov khoom siv raug xaiv, cov ntaub ntawv saum toj no tau txiav txim siab.
Tam sim no, kev tsim thiab tsim cov khoom siv fluorescent quantitative PCR cov khoom siv yog cov cuab yeej paub tab.Tsuav koj tsis xaiv cov tuam txhab lag luam phem heev, qhov tshwm sim ntawm cov teeb meem tsis yog siab, tab sis peb tseem xav qhia rau koj ob peb lub ntsiab lus:
Hot-start Taq enzyme:Qhov tseem ceeb tshaj plaws ntawm PCR yog qhov kub pib Taq enzyme.Cov enzymes kub-pib nyob rau hauv kev ua lag luam feem ntau muab faib ua ob hom, ib qho yog cov tshuaj hloov pauv kub-pib enzyme (koj tuaj yeem xav tias nws yog paraffin embedding), thiab lwm yam yog kub-pib enzyme rau kev hloov kho antibody (antigen-antibody khi).Kev hloov kho tshuaj yog ib txoj hauv kev thaum ntxov ntawm cov enzymes kub pib.Thaum muaj qhov kub thiab txias, lub enzyme yuav tso tawm nws cov haujlwm.Antibody-modified hot-start enzyme siv cov txheej txheem lom neeg los thaiv kev ua haujlwm ntawm enzyme.Thaum muaj qhov kub thiab txias, cov tshuaj tiv thaiv yuav raug denatured thiab inactivated raws li cov protein, thiab cov enzyme kev ua si yuav raug coj los ua si.
Txawm li cas los xij, qhov kev siv no yog dab tsi?Qhov no yog qhov teeb meem, qhov kev tso tawm ntawm cov tshuaj tiv thaiv-hloov enzymes sai dua li cov tshuaj hloov kho enzymes, yog li hais txog kev nkag siab zoo, cov tshuaj tiv thaiv-hloov enzymes muaj qhov zoo me ntsis, yog li tsis muaj cov tshuaj hloov pauv enzymes hauv cov khoom siv hauv khw.Yog tias muaj, ces qhov chaw tsim khoom siv thev naus laus zis tseem nyob hauv lub sijhawm xyoo txhiab xyoo.
Magnesium ion concentration:Magnesium ion concentration tseem ceeb heev hauv cov tshuaj tiv thaiv PCR.Tsim nyog magnesium ion concentration tuaj yeem txhawb kev tso tawm ntawm Taq enzyme kev ua.Yog tias qhov concentration tsawg dhau, cov haujlwm enzyme yuav raug txo kom tsawg;Yog hais tias qhov concentration siab dhau lawm, lub enzyme-catalyzed tsis-specific amplification yuav txhim kho.Cov concentration ntawm magnesium ions tseem yuav cuam tshuam rau kev sib tsoo ntawm cov primers, qhov kub thiab txias ntawm cov khoom siv PCR, yog li cuam tshuam rau cov txiaj ntsig ntawm cov khoom tawg.Qhov concentration ntawm magnesium ions feem ntau yog tswj ntawm 25mM.Tau kawg, rau cov khoom siv zoo, qhov concentration ntawm magnesium ions yuav tsum tau tswj kom zoo.Qee cov tub lag luam ntxiv cov tshuaj magnesium ion chelating tus neeg sawv cev rau cov reagent, uas tuaj yeem ua tiav cov txiaj ntsig ntawm kev hloov pauv tsis siv neeg ntawm magnesium ion concentration.
Fluorescent dye concentration:Fluorescent dye, uas yog SYBR Green peb feem ntau siv, feem ntau tsim cov fluorescence los ntawm kev khi rau qhov me me ntawm ob-stranded DNA, vim hais tias kev khi ntawm cov xim rau ob-stranded DNA tsis yog tshwj xeeb, uas yog hais tias ntev npaum li ob-stranded DNA tau ua ke nrog nws, fluorescence yuav tshwm sim hauv cov txheej txheem ntawm cov txheej txheem thiab cov txheej txheem sib xyaw ua ke. teeb liab.
PS: Vim nws lub teeb-rhiab heev, cov khoom ntawm lub khw feem ntau ntim rau hauv cov xim av opaque centrifuge raj (raws li qhia hauv daim duab hauv qab no).Txawm li cas los xij, qhov no yuav ntsib teeb meem.Nws yog ib qho nyuaj rau pom seb cov kua yog nqus thaum kuaj.Nyob rau hauv qhov kev hwm no, Qingke yog qhov tseeb cov neeg siv khoom zoo tshaj plaws (raws li qhia hauv daim duab hauv qab no), thiab lub raj pob tshab tau ntim rau hauv lub hnab opaque tin.Tom qab ntawd muab tso rau hauv ib lub hnab tin, coj mus rau hauv tus account qhov yooj yim ntawm kev zam lub teeb thiab sampling.Koj yuav tsum xaiv tus naj npawb ntawm cov khoom tsim nyog.TSE204 yog qhov muaj txiaj ntsig zoo heev, uas ua rau kuv xav cog nyom.
Qhov concentration ntawm fluorescent dye kuj tseem ceeb heev.Yog hais tias qhov concentration tsawg dhau lawm, lub amplification nkhaus yuav tsis nce mus rau theem tom ntej thiab tsis zoo tag nrho;Yog tias qhov concentration siab dhau lawm, nws yuav ua rau muaj suab nrov cuam tshuam.Txij li thaum cov fluorescent ntau PCR feem ntau yog nyob ntawm tus nqi CT, yog tias qhov concentration ntawm cov fluorescent dye tsis kho kom raug, qhov qis qis yog qhov zoo tshaj qhov siab.Tau kawg, qhov tsim nyog dye concentration yog qhov zoo tshaj plaws.
ROX: ROX dyes yog siv los kho cov teeb liab fluorescence kom zoo.Qee cov tuam txhab lag luam xav tau calibration, thaum lwm tus tsis ua.Piv txwv li, kev siv Thermo Fisher Scientific's Real Time PCR amplification instrument feem ntau yuav tsum tau calibration, suav nrog 7300, 7500, 7500Fast, StepOnePlus, thiab lwm yam. Cov lus qhia dav dav yuav piav qhia txog nws.
Foregene's qPCR Mix kuj muaj ROX dye, uas yooj yim rau kev siv ntau yam qauv.
Tsis muaj zog hydrogen bond kho: Kev kho mob ntawm cov tsis muaj zog hydrogen bonds yog ib qho teeb meem tseem ceeb.Tsis muaj dab tsi tau nyeem phau ntawv qhia ntawm ntau cov khoom siv, tab sis tsis muaj leej twg hais txog lub ntsiab lus no.Qhov tseeb, nws tseem ceeb heev.Kev sib xyaw ntawm cov hauv paus feem ntau yog nyob ntawm lub zog ntawm hydrogen bonds.Muaj zog hydrogen bonds yog ib txwm amplification, thiab tsis muaj zog hydrogen bonds ua rau tsis muaj ib qho amplification.Yog tias tsis muaj zog hydrogen bonds tsis tuaj yeem tshem tawm tau zoo, tsis muaj kev nthuav dav tshwj xeeb tsis tuaj yeem zam.Nyob rau hauv lub Scope ntawm tus sau, tsuas yog ob peb lub tuam txhab tau pom qhov teeb meem no.Thaum koj yuav cov khoom siv, koj tuaj yeem xa mus rau seb koj puas tau txiav txim siab txog qhov kev daws teeb meem no rau cov khoom siv uas koj xav xaiv.
Cov tshuaj tiv thaiv ntim: Lub tshuab 20-50ul feem ntau yog siv, thiab cov ntim me me yuav ua rau yuam kev.Feem ntau hais lus, cov lus qhia cov khoom siv yuav pom zoo siv cov tshuaj tiv thaiv PCR.Tsis txhob ntse thiab siv cov ntim me me kom txuag tau cov nqi.lub hom phiaj ntawm.Lub ntim tau pom zoo los ntawm cov tub lag luam tau raug sim tiag tiag, thiab nws yuav yog tias lawv tsis tuaj yeem daws qhov teeb meem ntawm qhov yuam kev los ntawm cov ntim me me.
2. Cov chaw tsim khoom thiab cov ntawv xov tooj ntawm lub raj phaj
Txhua tus paub lub hauv paus ntsiab lus ntawm fluorescent quantitative PCR.Kev sau fluorescence feem ntau yog ua los ntawm PCR lub kaus mom.Thaum xaiv PCR consumables, xyuam xim rau ob lub ntsiab lus: zoo lub teeb kis tau tus mob thiab haum rau lub ntsuas.Feem ntau hais lus, cov laug cam thiab cov hlab ntawm cov khoom lag luam tseem ceeb yog qhov zoo, tab sis koj yuav tsum xaiv kom zoo rau kev hloov kho, txwv tsis pub koj yuav tsis tuaj yeem siv qhov ntsuas.
4. Cov kev paub zoo tshaj plaws
MIQE (8)—qPCR validation
Qhov no yog qhov tseem ceeb tshaj plaws ntawm qPCR!Muaj ntau tus phab ej tau poob rau hauv cov xuab zeb ntawm no.Tau kawg, nws tseem ua tau tias koj muaj hmoo thiab cov noob uas koj tau kawm yog qhov yooj yim, yog li koj ntab hla lub qhov tsua dej khov raws cua.Cov ntaub ntawv pov thawj ntawm qPCR yog npaj los ntsuas qhov kev ntseeg tau ntawm cov ntaub ntawv.Peb sau cov ntaub ntawv pov thawj tsim nyog raws li hauv qab no:
1.Specificity xeem
Qhov tshwj xeeb ntawm lub hom phiaj gene amplification yog kuaj los ntawm kev tshuaj xyuas seb daim duab electrophoresis puas yog ib pawg;kev txheeb xyuas qhov sib npaug;melting nkhaus kom pom tias daim ntawv qhia ncov yog ib leeg;enzyme digestion pov thawj thiab lwm txoj kev.
Ntawm no, peb tsom rau tnws tsom xam ntawm cov tsis tshwj xeeb amplification siv txoj kev ntawm melting nkhaus.Feem ntau hais lus, thaum peb tsim cov primers, qhov loj ntawm cov khoom tawg yog yuav tsum tau nyob rau hauv thaj tsam ntawm 80-200bp, uas ua rau lub melting kub ntawm cov khoom PCR 80-85 ° C ntau yam.Yog li ntawd, yog hais tias muaj ntau yam ncov, yuav tsum muaj lwm yam khoom uas tsis tshwj xeeb amplification;yog tias lub ncov tshwm hauv qab 80 ° C, nws feem ntau suav tias yog primer dimer;Yog hais tias lub ncov tshwm saum toj 85 ° C, nws feem ntau suav hais tias yog DNA paug lossis ntau dua Nonspecific amplification ntawm cov tawg loj.
Lus Cim: Qee zaum tsuas muaj ib lub ncov ntawm 80 ° C.Lub sijhawm no, lub tswv yim no yuav tsum tau ua raws li.Nws yuav zoo li tias cov txiaj ntsig kev nthuav dav yog tag nrho cov primer dimers.
Li qub melting nkhaus (ib lub ncov uas tsis muaj qhov tshwj xeeb amplification)
Teeb meem melting nkhaus (tsis tshwj xeeb amplification ntawm spurious peaks)
【Case Analysis】
Muaj lub ncov loj, tab sis primer dimer yog qhov hnyav
Qhov nkhaus ntawm ib lub ncov melting nyob rau hauv daim duab hauv qab no tuaj yeem yooj yim dag koj ob lub qhov muag, xav tias nws yog qhov kev sim zoo meej, tab sis qhov tshwm sim yog qhov tsis ncaj ncees lawm.Nyob rau lub sijhawm no, peb yuav tsum tau saib qhov kub ntawm melting.Lub ncov kub yog qis dua 80 ° C, uas yog tag nrho primer-dimer.
Tsis muaj lub hom phiaj tawg, tag nrho cov primer dimers
Ntawm no, kuv tus tij laug nres tsis tau.Daim duab hauv qab no yog ib daim duab thaij nrog lub xov tooj ntawm tes xa tuaj rau kuv los ntawm ib tug scumbag.Cov reagents nws siv yog tag nrho cov npe siv hauv kev lag luam.Nws hloov los ntawm ib lub npe T-prefix mus rau lwm lub npe T-prefix.Kuv xav tias koj twb twv nws lawm.Tus scumbag quaj rau kuv: “Cov tshuaj reagent siv hauv thawj daim duab yog qhov zoo heev, thiab lub ncov yog ib leeg.Tom qab ntawd, tom qab siv cov tshuaj reagent koj pom zoo, nws yuav zoo li daim duab thib ob, nrog cov peaks sib xyaw.Koj ua rau kuv nyuaj siab.“
Cais ob daim duab.Thaum xub thawj siab ib muag, ib tug muaj ib tug ncov, thiab lwm yam muaj ob lub ncov.Tsis muaj tseeb, ib lub ncov yog qhov zoo.Puas muaj tseeb?
Phem tshaj Dou E, yog kuv tso ob daim duab hauv qab no koj yuav nkag siab tam sim ntawd.Qhov tseeb, peb tau yooj yim tuag tes tuag taw los ntawm cov duab zoo li no.Tom qab ua tib zoo tshuaj xyuas, peb pom tias: lub ncov ntawm thawj daim duab yog nyob rau ntawm 75 ° C, uas yog kiag li primer dimer;lub ncov ntawm daim duab thib ob tshwm ntawm 75 ° C thiab 82 ° C, yam tsawg kawg yog cov khoom tshwm.
Cov duab ntawm kev tawm tswv yim los ntawm cov tub ntxhais kawm
Yog li qhov teeb meem tseem ceeb tsis yog qhov teeb meem ntawm reagents, tab sis qhov teeb meem ntawm primer tsim.Nyob rau tib lub sijhawm, nws kuj ua pov thawj tias qee lub npe loj tsis yog hlau zoo, thiab nws kuj ua pov thawj qhov kuv tus tij laug tau hais ua ntej: Nws tsis yog lub npe reagent uas txhawb koj tsab xov xwm.Nws yog koj tsab xov xwm uas propped li hom reagents.Cia li xav txog tej yam, yog hais tias tus scumbag tsis hloov cov reagents, cov ntaub ntawv tsis ncaj ncees lawm yuav raug xa mus rau hauv phau ntawv journal, thiab dab tsi yuav tshwm sim yog ib qho xwm txheej.
2. Ct tus nqi ntawm kev tswj tsis pub muaj
Tsis txhob piav, yog tias qhov kev tswj tsis pub muaj tus nqi Ct, puas yog tsis muaj kuab paug?Txawm li cas los xij, koj tseem yuav tsum nkag siab qhov twg kev tswj hwm dawb muaj tus nqi Ct.Yog tias nws yog NTC, nws txhais tau hais tias muaj DNA txawv teb chaws xws li reagent paug.Yog tias nws yog NRT, nws txhais tau hais tias RNA rho tawm muaj DNA paug.
3. Txheem nkhaus
Nrog rau txoj kab nqes thiab kev suav cov mis, PCR efficiency tuaj yeem xam los ntawm cov qauv.Ib qho kev sim zoo tshaj plaws yuav tsum muaj txoj kab nqes ntawm tus qauv nkhaus mus rau 3.32, thiab R² mus txog 0.9999.
4. Linear dynamic ntau yam
Lub dynamic ntau ntawm cov tshuaj tiv thaiv yog linear.Raws li tus qauv siv los tsim cov qauv nkhaus, qhov ntau yam yuav tsum muaj tsawg kawg yog 5 concentration gradients, thiab xyuam xim rau qhov kev hloov ntawm Ct qhov tseem ceeb ntawm high concentration gradients thiab low concentration gradients.
5. Kev txheeb xyuas qhov tseeb
Kev hloov pauv hauv qPCR cov txiaj ntsig, uas yog, tsis zoo rov ua dua, uas yog, tsis zoo precision, yog tshwm sim los ntawm ntau yam, suav nrog qhov kub thiab txias, concentration, thiab kev ua haujlwm.qPCR precision feem ntau dhau los tswj tau tsawg dua li cov ntawv luam txo qis.Qhov zoo tshaj plaws nyob rau hauv-kev sim sib txawv, qhov kev hloov pauv no yuav tsum sib txawv ntawm kev sib txawv ntawm kev lom neeg, thiab cov khoom siv roj ntsha tuaj yeem hais ncaj qha txog qhov sib txawv ntawm cov lej hauv qPCR cov txiaj ntsig ntawm pab pawg lossis kev kho mob.Hauv tshwj xeeb rau kev kuaj mob, qhov zoo tshaj plaws inter-assay precision (repeatability) thoob plaws cov chaw thiab cov neeg ua haujlwm yuav tsum tau tshaj tawm.
6. Kev txheeb xyuas efficiency thiab LOD (hauv multiplex qPCR)
LOD yog qhov qis tshaj plaws ntawm 95% ntawm cov qauv kuaj pom zoo.Hauv lwm lo lus, qhov concentration ntawm LOD muaj nyob rau hauv ib txheej ntawm lub hom phiaj gene replicates yuav tsum tsis txhob ntau tshaj 5% ntawm qhov ua tsis tau tejyam tshwm sim.Thaum ua multiplex qPCR tsom xam, tshwj xeeb tshaj yog rau tib lub sij hawm nrhiav kom tau cov kev hloov pauv ntawm cov ntsiab lus los yog polymorphisms, multiplex qPCR yuav tsum tau muab pov thawj tias qhov tseeb ntawm ntau lub hom phiaj fragments tsis cuam tshuam rau tib lub raj, ntau qhov kev tshawb nrhiav thiab ib lub raj nrhiav kom tau Efficiency thiab LOD yuav tsum zoo ib yam.Tshwj xeeb tshaj yog thaum lub hom phiaj siab siab siab thiab qis qis lub hom phiaj cov noob yog ib txhij ua kom nthuav dav, qhov teeb meem no yuav tsum tau them sai sai rau.
Teeb meem thiab kev daws teeb meemFeem ntau hais lus, cov teeb meem feem ntau ntsib hauv qPCR debugging tsom rau cov hauv qab no:
· nonspecific amplification
· Kev xaiv nyuaj ntawm primer concentration thiab teeb meem nrog primer-dimers
· Cov kub annealing tsis raug
· Cov qauv thib ob cuam tshuam rau kev ua kom muaj zog
nonspecific amplification
non-specific amplificationtshwm sim, nws feem ntau txiav txim siab seb tus primer tsim tsis haum, tab sis yog tias koj tsis maj nrawm hloov cov primers, koj tuaj yeem sim cov hauv qab no ua ntej (lub hauv paus ntsiab lus tseem txuas nrog):
· Ua kom qhov kub thiab txias - sim ua kom tsis muaj zog hydrogen bonds tswj tsis tau;
· Shorten annealing thiab elongation lub sij hawm - txo lub sij hawm ntawm qaug zog hydrogen bonds;
· Txo cov primer concentration - txo lub caij nyoog ntawm kev khi ntawm redundant primers thiab cov cheeb tsam uas tsis yog hom phiaj;
Tsawg amplification efficiency
Qhov xwm txheej tsis sib xws rau qhov tsis muaj kev nthuav dav - qhov ua kom pom kev ua haujlwm tsis zoo, thiab kev ntsuas los cuam tshuam nrog kev ua kom tsis muaj zog yog qhov sib txawv:
· Ntev lub sij hawm annealing thiab elongation;
· Hloov mus rau peb-kauj ruam PCR thiab txo qhov kub ntawm annealing;
· Ua kom cov primer concentration;
Ps: Ntau tus tub ntxhais kawm tiav uas yug hauv 90s tsis kam kawm txog kev sim debug, thiab cia siab tias cov khoom siv tuaj yeem daws qhov teeb meem tag nrho (yog tias koj xav mus rau lub tuam txhab reagent los ua kev tshawb fawb thiab kev loj hlob tom qab kawm tiav), qhov tseeb, cov reagent manufacturers kuj xav li no, kuv vam tias nws yog ib tug neeg ruam nws yuav siv tau thaum koj tau txais nws, yog li ntawd reagent manufacturers daws cov teeb meem tsis muaj zog ntawm H. -bond absorption yam.Yuav kom daws tau qhov teeb meem yooj yim, cov neeg ruam tseem yuav tsum tau nyeem cov lus qhia ntawm lub tuam txhab reagent kom pom tias muaj ib qho tseem ceeb uas absorbs tsis muaj zog hydrogen bonds.
Kev xaiv nyuaj ntawm primer concentration thiab teeb meem nrog primer-dimers
Txoj kev 1: Feem ntau hais lus, cov lus qhia cov khoom siv rau qPCR muaj cov txheej txheem pom zoo thiab pom zoo primer concentrations.
Txoj kev 2: Debugging los ntawm kev teeb tsa cov primer concentration gradient.Daim duab hauv qab no yog nyiag los ntawm ib lub tuam txhab los piav qhia.Daim duab hauv qab no qhia tau hais tias fluorescence quantitative tau ua nrog peb primer concentration gradients (100nM, 250nM, 500nM) thiab plaub template concentration gradients (0.1ng, 1ng, 10ng, 100ng).Tus nqi Ct ntawm cov txiaj ntsig kev sim yog npaj raws li hauv qab no:
Primer Concentration Selection Concatenate txhua primer concentration rau hauv ib kab raws li hauv qab no:
Qhov kev xaiv ntawm primer concentration yog pom tseeb, txoj kev sib raug zoo ntawm primer concentration ntawm 100nM thiab 250nM yog qhov zoo dua, thiab linear kev sib raug zoo ntawm primer concentration ntawm 500nM yog qhov tsis zoo.Hauv 100nM thiab 250nM, Ct tus nqi ntawm 250nM kuj me me, yog li qhov zoo tshaj plaws primer concentration yog 250nM.Feem ntau hnyav primer-dimers tuaj yeem pom nyob rau hauv qhov nkhaus melting.Yuav ua li cas yog tias cov primers tsim tsis tuaj yeem zam primer-dimers?
Txoj kev 3: Txo tus nqi ntawm primers thiab nce qhov kub thiab txias (tsis tas yuav piav qhia).
Tus nqi empirical ntawm annealing kub yog 60 ° C.Yog tias koj tsis paub meej, yuav ua li cas xaiv qhov ntsuas kub zoo dua?Cov lus teb yog tib yam li kev xaiv ntawm primer concentration -gradient xeem.Siv ib daim duab los ntawm Bio-rad tuam txhab los qhia txog qhov teeb meem.Rau kev nthuav dav ntawm qee lub hom phiaj, teem yim qhov kub thiab txias gradients, txhua tus nrog peb rov ua dua, thiab qhov tau txais kev nthuav dav yog raws li hauv qab no:
annealing kub xaiv:
· 70 ° C, 69 ° C - Yeej, cov primers tsis tuaj yeem ua ke, yog li tsis muaj kev nthuav dav.
· 67.3 ° C - Muaj me me ntawm amplification thaum pib, thiab tus nqi Ct kuj loj.
· 64.5°C — Tus nqi Ct txo.
· Ntawm 60.7 ° C, 58.0 ° C, 56.2 ° C, thiab 55.0 ° C, Ct qhov tseem ceeb tau zoo nyob ruaj khov, tab sis qhov kawg fluorescence qhov tseem ceeb sib txawv.
Yuav xaiv li cas?Lub hauv paus ntsiab lus: Thawj lub hauv paus ntsiab lus yog tus nqi Ct siab dua.Rau tib tus nqi Ct, xaiv qhov kub siab dua annealing kom tsis txhob dimerization thiab tsis yog qhov tshwj xeeb amplification.Txawm hais tias muaj tus nqi fluorescence ntau dua ntawm 55 ° C, tej zaum yuav muaj cov dimers lossis tsis muaj kev nthuav dav hauv nws.
Tab sis yog tias koj ntse li koj, koj yuav twv yuav raug hu xav tias: Kev hais lus zoo, yog tias cov tshuaj tiv thaiv PCR yog qhov tshwj xeeb, tsuav yog cov primer concentration tshaj qhov tsawg kawg nkaus yuav tsum tau, cov ntsiab lus siab thiab qis yuav tsum tsis muaj txiaj ntsig, ib yam li fluorescent dyes thiab dNTPs.Tseeb tiag, tsuav yog qhov kub thiab txias annealing yog optimized kom zoo, cov nyhuv ntawm primer concentration ntawm Ct tus nqi yuav ib txwm txo.
Qhov kub annealing yog optimized kom zoo, thiab cov nyhuv ntawm primer concentration ntawm CT yuav raug txo
Secondary qauv cuam tshuam rau amplification efficiency
Cia peb nqa daim duab los ntawm Bio-rad los qhia txog qhov teeb meem.Nws kuj tsim qhov kub thiab txias gradient los nthuav cov noob nrog cov qauv thib ob.
Secondary qauv tshwm sim
Nws tuaj yeem pom tau tias thaum qhov ntsuas kub qis qis, cov khoom pib tshwm sim thiab tus nqi Ct txav mus rau tom ntej, mus txog qhov tsawg kawg nkaus tus nqi ntawm 60.7 ° C, thiab tom qab ntawd raws li qhov ntsuas kub qis qis, Ct tus nqi yuav loj dua.Hloov pauv, raws li qhov kub thiab txias nce, cov qauv thib ob qhib thiab qhov kev ua kom zoo dua ntxiv.Tom qab ncav cuag qhov kub thiab txias, nce qhov kub tsis tuaj yeem txhim kho qhov kev ua kom zoo dua qub.Vim tias cov primers tsis tuaj yeem ua ke ruaj khov rau lub sijhawm no.Yog li ntawd,nrhiav qhov kub thiab txias nrog tus nqi qis tshaj Ct, uas yog qhov zoo tshaj plaws kub rau amplifying lub thib ob qauv template!Tau kawg, cov neeg ruam ntse yuav tsum paub tias yog tias nws tsis tsim nyog, nws yog qhov zoo tshaj plaws los hloov cov primers thiab zam cov cheeb tsam theem nrab.
5. Daim ntawv thov qib
MIQE-Data Analysis
Cov ntaub ntawv tsom xam feem ntau yog muab los ntawm fluorescent quantitative PCR ntsuas.Nyob rau hauv tsab xov xwm dhau los, ntau cov ntaub ntawv tsom xam ua haujlwm tau ua tiav, xws li kev tswj hwm dawb huv, uas tau piav qhia hauv kev tsim qauv ntawm kev sim.Cov noob siv sab hauv, cov lej rov qab, thiab lwm yam tau qhia meej., ntawm no peb feem ntau piav qhia txog kev thov qPCR.
qPCR tau siv dav, thiab kev sim tshuaj ntsuam xyuas thiab kuaj nucleic acid yog qhov feem ntau siv cov xwm txheej.
kom muaj nuj nqis
Log (thawj concentration) muaj ib tug linear kev sib raug zoo nrog tus naj npawb ntawm cycles.Ib qho qauv nkhaus tuaj yeem kos los ntawm tus qauv nrog tus lej luam thawj zaug, uas yog, qhov kev sib raug zoo ntawm cov tshuaj tiv thaiv amplification tuaj yeem tau txais.Raws li tus nqi Ct ntawm tus qauv, qhov concentration hauv cov qauv tuaj yeem suav tau.Tus nqi ntawm cov qauv yuav suav nrog.
Tus Txheej Txheem Kev Ua Kom Muaj Zog
Kev ntsuas qhov tseeb yuav tsum yog raws li tus qauv nkhaus.Txhawm rau ua tus qauv nkhaus, yuav tsum muaj tus qauv.Feem ntau, tus qauv yog plasmid tau los ntawm cloning lub hom phiaj gene.Vim li cas nws yog plasmid?Vim hais tias ncig plasmid DNA yog qhov ruaj khov tshaj plaws.Dilute cov khoom txheem hauv 5 mus rau 6 gradients raws li qhov sib piv ob npaug (10-fold dilution), thiab xyuam xim rau qhov sib xws thaum diluting.Cia tus nqi Ct poob ntawm 15-30.
Txuj kev npaj
Nyob rau tib lub sijhawm, tus qauv yuav tsum tau kuaj yuav tsum tau diluted raws li (nco txog qhov dilution factor), thiab tus nqi Ct kuj tseem yuav poob ntawm 15-30.Cov khoom txheem + cov qauv yuav tsum tau kuaj yog muab tso rau hauv lub tshuab ua ke.Tom qab kev khiav, ib qho qauv nkhaus tau ua nrog cov khoom txheem, thiab cov qauv yuav tsum tau kuaj tau raug coj mus rau hauv tus qauv nkhaus los xam qhov concentration.
Tus kab mob siab B tus kab mob HBV yog ib qho tseem ceeb kom muaj nuj nqis, uas tuaj yeem xam tus kab mob tus lej hauv 1ml ntawm cov ntshav.
Kev suav cov lej luam
Sample concentration yuav tsum tau kuaj (ng / ul) = OD260 × 50ug / ml × dilution factor
Cov qauv molecular hnyav = tus naj npawb ntawm cov hauv paus × 324
Tus lej luam ntawm tus qauv yuav raug kuaj (cov ntawv luam / ul) = qhov concentration ntawm cov qauv yuav tsum tau kuaj / qhov hnyav molecular ntawm tus qauv × 6 × 1014
Txoj kev suav ntawm tus lej luam
Cov saum toj no yog txoj kev suav rau kev txiav txim kom muaj nuj nqis.Qhov no yog ib qho teeb meem kev ua lej uas tuaj yeem daws tau tom qab kawm tiav qib siab, thiab cov teeb meem lej feem ntau daws tau los ntawm khoos phis tawj.Yog koj tsis nkag siab, koj tuaj yeem tuaj sib tham.
txheeb ze kom muaj nuj nqis
Relative quantification yog siv nyob rau hauv kev tshawb fawb.Muaj pes tsawg tus kab mob muaj nyob rau hauv 1ml ntawm cov ntshav, thiab nws yog ib tug kab mob DNA, qhov no yog ib tug kuj txiav txim qhov tshwm sim: tus nqi ntawm cov ntshav yuav txiav txim tau, thiab DNA tus kab mob no kuj ruaj khov.Txawm li cas los xij, nws yog qhov nyuaj rau peb los sib piv cov lej ntawm cov ntawv luam ntawm qee cov noob hauv nplooj, vim tias nws nyuaj rau txiav txim siab qhov loj, qhov hnyav, thiab rhiab ntawm nplooj, tus nqi ntawm RNA rho tawm yog qhov nyuaj rau kev txiav txim siab, thiab qhov ua tau zoo ntawm kev hloov pauv rov qab kuj nyuaj rau kev txiav txim siab, uas yog hais tias, txhua kauj ruam yuav ua rau cov ntaub ntawv sim muaj kab thiab siv tsis tau.
Yog li ntawd, tus txheeb ze quantification yuav tsum qhia ib lub ntsiab:lub internal reference gene.
Hauv lwm lo lus, kev txheeb xyuas qhov txheeb ze yog qhov sib piv ntawm lub hom phiaj gene thiab lub hauv paus siv cov noob.Muab piv rau tib cov ntaub so ntswg thiab tib lub xovtooj ntawm tes, kev cuam tshuam ntawm tus qauv loj, RNA rho tawm tus nqi, rov qab ua kom zoo dua qub, thiab PCR efficiency yog me me.Vim yog cov qauv me me, ob qho tib si siv cov noob thiab cov hom phiaj cov noob tau raug txo qis.Qhov no yog vim li cas peb tau hais txog kev tsis sib xws thiab kev ruaj ntseg ua ntej.
Internal reference noob yog feem ntautsev tu noob(cov noob khaws cia hauv tsev), uas xa mus rau ib chav kawm ntawm cov noob uas muaj kev ruaj ntseg hauv txhua lub hlwb, thiab lawv cov khoom tsim nyog los tswj cov kev ua ub no ntawm cov hlwb.
Tsis txhob nkim lub tswvyim no.Cov noob hauv tsev yog cov ntsiab lus ua haujlwm lom neeg, thaum cov noob siv sab hauv yog cov ntsiab lus kev sim.Cov noob hauv tsev yuav tsum dhau qhov kev lees paub ua ntej lawv tuaj yeem raug xaiv los ua cov noob siv sab hauv.
Piv txwv li, peb tau xaiv ntau lub tsev tu noob hauv daim duab hauv qab no los ntsuas lawv cov theem qhia hauv cov ntaub so ntswg sib txawv, thiab pom tias cov qib qhia ntawm β-2-microglobulin txawv ntawm lwm cov noob, yog li lawv tsis tuaj yeem siv los ua cov noob siv sab hauv.
Tom qab nkag siab txog kev kho qhov ua haujlwm ntawm lub hauv paus siv cov noob, ob lub algorithms tau muab los ntawm kev qhia txog cov gene hauv cov ntaub ntawv.
· ob npaug qauv nkhaus txoj kev
· 2 – △△ Ct method (CT value comparison method)
Yog tias koj xav kawm txog hom kab mob thiab cov noob caj noob ces, thov tso tseg qhov kev tshawb fawb ntawm algorithms thiab siv cov qauv ncaj qha, lossis siv cov tshuab ncaj qha;yog tias koj yog ib tug txiv neej ncaj hauv kev ua lej thiab engineering, thov koj xav dawb.
ob txheej txheem nkhaus txoj kev
Txheeb xyuas cov hom phiaj gene thiab tsev tu noob ntawm cov qauv tswj thiab cov qauv yuav tsum tau kuaj los ntawm tus qauv nkhaus, thiab tom qab ntawd xam tus nqi txheeb ze raws li cov qauv kev suav, uas yog cov txheeb ze qhia theem.
Qhov zoo: yooj yim tsom xam, kuj yooj yim sim optimization
Disadvantage: Rau txhua tus noob, txhua qhov kev sim yuav tsum ua kom muaj tus qauv nkhaus
Daim Ntawv Thov: Ib qho ntawm ob feem ntau siv thiab pom tau tias muaj pes tsawg tus txheeb ze txoj hauv kev kawm txog kev tswj cov noob caj noob ces
Cov mis yog raws li nram no:
Piv txwv li no:
Xam tus txheeb ze npaum li cas raws li qhov tshwm sim ntau
2 – △△ Ct method (CT value comparison method)
Qhov zoo: Tsis tas yuav ua tus qauv nkhaus
Disadvantages: Nws yog assumed tias lub amplification efficiency yog ze rau 100%;tus qauv sib txawv yog <5%, thiab tus qauv nkhaus thiab qhov ua tau zoo ntawm txhua qhov kev nthuav dav yog suav tias yog qhov sib xws;qhov optimization ntawm kev sim tej yam kev mob yog ntau nyuaj.
Daim Ntawv Thov: Ib qho ntawm ob feem ntau siv thiab pom tau tias muaj pes tsawg tus txheeb ze txoj hauv kev kawm txog kev tswj cov noob caj noob ces
Tau kawg, qhov kev ua kom zoo tshaj plaws feem ntau tsis tuaj yeem ua tau zoo kawg nkaus 1. Kev kho txoj kev: Yog tias peb paub tias lub hom phiaj gene thiab cov neeg siv cov noob muaj tib qhov kev ua kom zoo dua qub, tab sis qhov kev ua kom zoo dua qub tsis sib npaug rau 1, ces 2－△△ Ct tuaj yeem kho tau raws li: (1 + E) －△△ Ct 5 yog qhov ua tau zoo, piv txwv li 0. rau 1.95－△△ Ct
Txog tam sim no, cov ntsiab lus hais txog fluorescent quantitative PCR tau los xaus.
Post lub sij hawm: Apr-06-2023
