Tus nqi Ct yog daim ntawv nthuav qhia tseem ceeb tshaj plaws ntawm fluorescent quantitative PCR.Nws yog siv los xam cov gene qhia qhov sib txawv los yog gene luam tus lej.Yog li dab tsi yog tus nqi Ct ntawm fluorescence quantification suav hais tias tsim nyog?Yuav ua li cas kom paub meej qhov zoo ntawm Ct tus nqi?
Tus nqi Ct yog dab tsi?
Thaum lub sij hawm qPCR amplification txheej txheem, tus naj npawb ntawm amplification cycles (Cycle Threshold) thaum lub teeb liab fluorescence ntawm cov khoom amplified mus txog lub teeb fluorescence pib.C stands rau Cycle thiab T stands rau Threshold.Cias muab tso, tus nqi Ct yog tus naj npawb ntawm cov voj voog sib raug rau thaum pib cov qauv kev nthuav dav mus txog qee yam khoom hauv qPCR.Lub npe hu ua "ib yam khoom lag luam" yuav piav qhia ntxiv tom qab.
Tus nqi Ct ua li cas?
1.Kev sib raug zoo ntawm exponential amplification, tus nqi template thiab tus nqi Ct
Qhov zoo tshaj plaws, cov noob hauv qPCR tau sau los ntawm kev nthuav dav ntxiv tom qab qee lub voj voog.Kev sib raug zoo ntawm tus naj npawb ntawm amplification cycles thiab tus nqi ntawm cov khoom yog: Amplified khoom nqi = thawj tus qauv nqi × (1+En) cycles naj npawb.Txawm li cas los xij, qPCR cov tshuaj tiv thaiv tsis yog nyob rau hauv qhov xwm txheej zoo tagnrho.Thaum tus nqi ntawm cov khoom lag luam tau nce mus txog "qee yam khoom lag luam", tus naj npawb ntawm cov voj voog ntawm lub sijhawm no yog tus nqi Ct, thiab nws yog nyob rau lub sijhawm nthuav dav.Kev sib raug zoo ntawm tus nqi Ct thiab tus nqi pib tus qauv: Muaj kev sib raug zoo ntawm tus nqi Ct ntawm tus qauv thiab tus logarithm ntawm tus lej pib ntawm tus qauv.Qhov siab dua qhov pib template concentration, qhov tsawg dua tus nqi Ct;qhov qis dua qhov pib template concentration, qhov loj dua tus nqi Ct.
2.Amplification nkhaus, fluorescence pib thiab tej yam khoom PCR
Tus nqi ntawm qPCR amplification khoom yog qhia ncaj qha nyob rau hauv daim ntawv ntawm fluorescent teeb liab, uas yog, lub amplification nkhaus.Nyob rau theem pib ntawm PCR, qhov kev nthuav dav yog nyob rau hauv cov xwm txheej zoo tagnrho, cov voj voog me me, cov khoom ntim me me, thiab qib ntawm fluorescence tsis tuaj yeem pom meej meej ntawm cov fluorescence keeb kwm yav dhau.Tom qab ntawd, fluorescence nce thiab nkag mus rau theem exponential.Tus nqi ntawm cov khoom PCR tuaj yeem kuaj pom ntawm qee qhov taw qhia thaum cov tshuaj tiv thaiv PCR tsuas yog nyob rau theem exponential, uas tuaj yeem siv los ua "ib qho ntawm cov khoom", thiab cov ntsiab lus pib ntawm tus qauv tuaj yeem txiav tawm ntawm qhov no.Yog li ntawd, fluorescence teeb liab siv sib txuam rau qee yam khoom yog qhov pib ntawm fluorescence.
Nyob rau hauv lub lig theem ntawm PCR, lub amplification nkhaus tsis qhia exponential amplification, thiab nkag mus rau lub linear theem thiab toj siab theem.
3.Reproducibility ntawm Ct tus nqi
Thaum lub voj voog PCR mus txog lub voj voog tus lej ntawm tus nqi Ct, nws nyuam qhuav nkag mus rau lub sijhawm exponential amplification tseeb.Lub sijhawm no, qhov yuam kev me me tsis tau nthuav dav, yog li qhov rov ua dua ntawm Ct tus nqi yog qhov zoo heev, uas yog, tib lub qauv yog amplified nyob rau lub sijhawm sib txawv lossis hauv cov raj sib txawv tib lub sijhawm.Amplification, qhov tau txais Ct tus nqi yog tas li.
1.Amplification efficiency En
PCR amplification efficiency yog hais txog qhov ua tau zoo uas cov polymerase hloov cov noob mus rau amplified rau hauv amplicon.Kev ua kom zoo dua qub thaum ib qho DNA molecule hloov mus rau hauv ob DNA molecules yog 100%.Amplification efficiency feem ntau qhia raws li En.Txhawm rau ua kom yooj yim rau kev txheeb xyuas cov khoom tom ntej, cov yam ntxwv uas cuam tshuam rau kev ua kom muaj zog amplification tau qhia luv luv.
| Kev cuam tshuam | piav qhia | Yuav txiav txim li cas? |
| A. PCR inhibitors | 1. Cov qauv DNA muaj cov tshuaj uas inhibit PCR cov tshuaj tiv thaiv, xws li cov proteins lossis tshuaj ntxuav tes.2. Lub cDNA tom qab kev hloov pauv rov qab muaj qhov siab ntawm cov qauv RNA lossis RT reagent Cheebtsam, uas tuaj yeem cuam tshuam cov tshuaj tiv thaiv PCR tom qab. | 1. Txawm tias muaj kuab paug tuaj yeem txiav txim los ntawm kev ntsuas qhov piv ntawm A260 / A280 thiab A260 / A230 lossis RNA electrophoresis.2. Txawm hais tias cDNA yog diluted raws li ib qho piv txwv tom qab thim rov qab. |
| B. Kev tsim primer tsis raug | Primers tsis ua haujlwm zoo | Xyuas cov primers rau primer-dimers los yog hairpins, tsis sib haum, thiab qee zaum ncua kev tsim intronic. |
| C. Kev tsim kho PCR tsis zoo | 1. Primers tsis tuaj yeem ua haujlwm zoo2. Kev tso tawm tsis txaus ntawm DNA polymerase 3. Lub sij hawm ntev kub siab DNA polymerase kev ua haujlwm txo qis | 1. Lub annealing kub yog siab tshaj TM tus nqi ntawm primer2. Pre-denaturation lub sij hawm luv heev 3. Lub sijhawm ntawm txhua theem ntawm cov txheej txheem tshuaj tiv thaiv ntev dhau lawm |
| D. Kev sib xyaw tsis txaus ntawm cov tshuaj reagents lossis pipetting yuam kev | Nyob rau hauv cov tshuaj tiv thaiv kab mob, lub zos concentration ntawm PCR tshuaj tiv thaiv Cheebtsam yog siab dhau los yog tsis sib xws, uas ua rau tsis-exponential amplification ntawm PCR amplification. | |
| E. Amplicon Ntev | Qhov ntev ntawm lub amplicon ntev dhau lawm, ntau tshaj 300bp, thiab qhov ua kom zoo dua qub yog qhov tsawg. | Txheeb xyuas tias qhov ntev amplicon nyob nruab nrab ntawm 80-300bp |
| F. Kev cuam tshuam ntawm qPCR reagents | Qhov concentration ntawm DNA polymerase nyob rau hauv lub reagent yog tsawg los yog cov concentration ntawm ions nyob rau hauv lub tsis yog optimized, ua rau lub Taq enzyme kev ua si tsis ncav cuag qhov siab tshaj plaws. | Kev txiav txim siab ntawm amplification efficiency los ntawm tus qauv nkhaus |
2.Range ntawm Ct tus nqi
Ct qhov tseem ceeb ntawm 15-35.Yog tias tus nqi Ct tsawg dua 15, nws raug txiav txim siab tias qhov kev nthuav dav yog nyob rau hauv thaj tsam ntawm lub sijhawm pib thiab qhov pib fluorescence tsis tau mus txog.Qhov zoo tshaj plaws, muaj kev sib raug zoo ntawm tus nqi Ct thiab lub logarithm ntawm thawj tus qauv ntawm tus qauv, uas yog, tus qauv nkhaus.Los ntawm tus qauv nkhaus, thaum lub amplification efficiency yog 100%, xam tus nqi Ct rau quantifying ib daim qauv ntawm cov noob yog nyob ib ncig ntawm 35. Yog hais tias nws yog ntau tshaj 35, thawj daim ntawv luam ntawm tus template yog theoretically tsawg tshaj li 1, uas yuav xam tau tias yog tsis muaj nuj nqis.
Rau qhov sib txawv ntawm cov noob Ct, vim qhov sib txawv ntawm cov noob luam tus lej thiab kev ua kom muaj txiaj ntsig zoo nyob rau hauv thawj tus qauv, nws yog ib qho tsim nyog los ua tus qauv nkhaus rau cov noob thiab suav cov kab kev tshawb pom ntawm cov noob.
3.Influencing yam ntawm tus nqi Ct
Los ntawm kev sib raug zoo ntawm tus naj npawb ntawm amplification cycles thiab tus nqi ntawm cov khoom: tus nqi ntawm cov khoom amplified = tus nqi ntawm thawj template × (1+En) lub voj voog tus lej, nws tuaj yeem pom tau tias nyob rau hauv cov xwm txheej zoo tagnrho, tus nqi ntawm cov qauv pib thiab En yuav muaj kev cuam tshuam tsis zoo rau Ct tus nqi cuam tshuam.Qhov txawv ntawm template zoo los yog amplification efficiency yuav ua rau tus nqi Ct loj los yog me me dhau.
4.Ct tus nqi loj dhau los yog tsawg dhau
Post lub sij hawm: Feb-22-2023
