1. Kev paub yooj yim (yog tias koj xav pom qhov kev sim, thov xa ncaj qha mus rau ntu thib ob)
Raws li cov tshuaj tiv thaiv ntawm PCR cov lus pom zoo, Lub sijhawm tiag tiag PCR feem ntau saib xyuas qhov kev hloov pauv ntawm cov khoom lag luam amplification hauv txhua lub voj voog ntawm PCR amplification cov tshuaj tiv thaiv hauv lub sijhawm los ntawm kev hloov pauv ntawm cov teeb liab fluorescence, thiab txheeb xyuas qhov ntau ntawm cov qauv pib los ntawm kev sib raug zoo ntawm tus nqi ct thiab tus qauv nkhaus.
Cov ntaub ntawv tshwj xeeb ntawm RT-PCR yoghauv paus, fluorescence pibthiabCt tus nqi.
| hauv paus: | Tus nqi fluorescence ntawm lub voj voog 3rd-15th yog lub hauv paus (baseline), uas yog tshwm sim los ntawm qhov yuam kev ntawm qhov ntsuas. |
| Qib (threshold): | Xa mus rau qhov txwv fluorescence nrhiav pom ntawm qhov tsim nyog nyob rau hauv cheeb tsam kev loj hlob exponential ntawm qhov kev nthuav dav nkhaus, feem ntau 10 npaug ntawm tus qauv sib txawv ntawm cov hauv paus. |
| CT tus nqi: | Nws yog tus lej ntawm PCR cycles thaum tus nqi fluorescence nyob rau hauv txhua lub raj tshuaj tiv thaiv mus txog qhov pib. Tus nqi Ct yog inversely proportional rau tus nqi pib template. |
Cov txheej txheem sau npe rau RT-PCR:
| txoj kev | qhov zoo | ua tsis tau | Scope ntawm daim ntawv thov |
| SYBR Green Ⅰ | Wide applicability, rhiab heev, pheej yig thiab yooj yim | Cov kev xav tau primer yog siab, nws yooj yim rau cov bands tsis tshwj xeeb | Nws yog qhov tsim nyog rau kev txheeb xyuas ntau yam ntawm cov hom phiaj, kev tshawb fawb ntawm cov noob qhia, thiab kev tshawb fawb txog cov tsiaj txhu thiab cov nroj tsuag transgenic recombinant. |
| TaqMan | Zoo meej thiab siab repeatability | Tus nqi siab thiab tsuas yog haum rau cov hom phiaj tshwj xeeb. | Kev tshawb nrhiav kab mob, kev tshawb fawb txog tshuaj tiv thaiv kab mob, tshuaj ntsuam xyuas kev siv tshuaj, kev kuaj mob caj ces. |
| molecular beacon | High tshwj xeeb, fluorescence, tsis tshua muaj keeb kwm yav dhau | Tus nqi yog siab, nws tsuas yog tsim nyog rau lub hom phiaj tshwj xeeb, tus tsim yog qhov nyuaj, thiab tus nqi siab. | Kev tshuaj ntsuam gene tshwj xeeb, SNP tsom xam |
2. Kev sim cov kauj ruam
2.1 Hais txog kev sim pab pawg- yuav tsum muaj ntau lub qhov dej hauv pawg, thiab yuav tsum muaj kev rov ua dua tshiab.
| ① | Kev tswj tsis pub muaj | Siv los txheeb xyuas cov xwm txheej ntawm cell loj hlob hauv kev sim |
| ② | Kev tswj tsis zoo siRNA (tsis yog siRNA ib ntus) | Ua kom pom qhov tshwj xeeb ntawm RNAi kev ua.siRNA tuaj yeem ua rau tsis muaj kev ntxhov siab tshwj xeeb ntawm qhov siab ntawm 200nM. |
| ③ | Transfection Reagent Control | Tsis suav cov tshuaj toxicity ntawm transfection reagent rau cov hlwb los yog cov nyhuv ntawm kev qhia ntawm lub hom phiaj gene |
| ④ | siRNA tiv thaiv lub hom phiaj gene | Khawb qhov kev qhia ntawm lub hom phiaj gene |
| ⑤ (yeem) | zoo siRNA | Siv los daws qhov teeb meem kev sim thiab teeb meem kev ua haujlwm |
| ⑥ (yeem) | fluorescent tswj siRNA | Kev ua haujlwm ntawm cell transfection tuaj yeem pom nrog lub tshuab tsom |
2.2 Cov ntsiab cai ntawm primer tsim
| Amplified fragment loj | Zoo dua ntawm 100-150bp |
| Primer Ntev | 18-25 Ib |
| GC cov ntsiab lus | 30% -70%, nyiam dua 45% -55% |
| Tm tus nqi | 58-60 ℃ |
| Ib ntus | Tsis txhob T / C txuas ntxiv;A/G nruam |
| 3 kawg sequence | Zam GC nplua nuj lossis AT nplua nuj;lub hauv paus davhlau ya nyob twg yog qhov zoo dua G lossis C;nws yog qhov zoo tshaj kom zam T |
| Complementarity | Tsis txhob sib xyaw ua ke ntawm ntau tshaj 3 lub hauv paus hauv cov primer lossis nruab nrab ntawm ob primers |
| Qhov tshwj xeeb | Siv blast search kom paub meej tias primer tshwj xeeb |
①SiRNA yog hom tshwj xeeb, thiab cov kab sib txawv ntawm ntau hom yuav txawv.
②SiRNA tau ntim rau hauv cov hmoov qhuav qhuav, uas tuaj yeem khaws cia ruaj khov rau 2-4 lub lis piam ntawm chav tsev kub.
2.3 Cov cuab yeej lossis cov tshuaj reagents uas yuav tsum tau npaj ua ntej
| Primer (Internal reference) | Nrog rau pem hauv ntej thiab thim rov qab ob |
| Primers (lub hom phiaj gene) | Nrog rau pem hauv ntej thiab thim rov qab ob |
| Target Si RNA (3 strips) | Feem ntau, lub tuam txhab yuav sib txuas 3 strips, thiab tom qab ntawd xaiv ib qho ntawm peb los ntawm RT-PCR |
| Transfection Kit | Lipo 2000 thiab lwm yam. |
| RNA Rapid Extraction Kit | Rau RNA extraction tom qab transfection |
| Rapid Reverse Transcription Kit | rau cDNA synthesis |
| PCR Amplification Kit | 2 × Super SYBR Ntsuab qPCR Master Mix |
2.4 Hais txog cov teeb meem uas yuav tsum tau them sai sai rau hauv cov kauj ruam kev sim tshwj xeeb:
①siRNA kev hloov pauv
1. Rau plating, koj tuaj yeem xaiv 24-zoo phaj, 12-zoo phaj lossis 6-zoo phaj (qhov nruab nrab RNA concentration tau npaj rau hauv txhua lub qhov dej ntawm 24-zoo phaj yog li 100-300 ng / uL), thiab qhov zoo tshaj plaws transfection ceev ntawm cov hlwb yog mus txog 60% -80% los yog li ntawd.
2. Cov kauj ruam hloov pauv thiab cov kev cai tshwj xeeb yog nruj me ntsis raws li cov lus qhia.
3. Tom qab hloov pauv, cov qauv tuaj yeem sau tau hauv 24-72 teev rau kev kuaj pom mRNA (RT-PCR) lossis kuaj pom cov protein hauv 48-96 teev (WB)
② RNA extraction txheej txheem
1. Tiv thaiv kom tsis txhob kis kab mob los ntawm exogenous enzymes.Nws feem ntau suav nrog hnav lub qhov ncauj qhov ntswg thiab hnab looj tes nruj me ntsis;siv sterilized pipette tswv yim thiab EP raj;Cov dej siv hauv kev sim yuav tsum yog RNase-Free.
2. Nws raug nquahu kom ua ob zaug raws li tau hais tseg hauv cov khoom siv nrawm nrawm, uas yuav txhim kho qhov purity thiab tawm los.
3. Cov kua khib nyiab yuav tsum tsis txhob kov RNA kem.
③ RNA kom muaj nuj nqis
Tom qab RNA raug rho tawm, nws tuaj yeem suav ncaj qha nrog Nanodrop, thiab qhov tsawg kawg nkaus nyeem ntawv tuaj yeem qis li 10ng / ul.
④ Cov txheej txheem thim rov qab
1. Vim yog qhov siab ntawm RT-qPCR, tsawg kawg 3 qhov sib npaug yuav tsum tau ua rau txhua tus qauv txhawm rau tiv thaiv cov Ct tom ntej los ntawm qhov sib txawv dhau los lossis SD kom loj dhau rau kev txheeb xyuas kev txheeb xyuas.
2. Tsis txhob khov thiab thaw Master sib tov ntau zaus.
3. Txhua lub raj / qhov yuav tsum tau hloov nrog lub taub tshiab!Tsis tas li siv tib lub pipette taub los ntxiv cov qauv!
4. Cov yeeb yaj kiab txuas nrog 96-zoo phaj tom qab ntxiv cov qauv yuav tsum tau smoothed nrog lub phaj.Nws yog qhov zoo tshaj rau centrifuge ua ntej muab tso rau hauv lub tshuab, kom cov kua ntawm lub raj phab ntsa tuaj yeem ntws thiab tshem tawm cov npuas.
⑤ Kev tsom xam nkhaus
| Tsis muaj lub sijhawm ntawm logarithmic loj hlob | Tejzaum nws yog siab concentration ntawm template |
| Tsis muaj CT tus nqi | Cov kauj ruam tsis raug rau kev kuaj pom cov teeb liab fluorescent; degradation ntawm primers lossis probes - nws txoj kev ncaj ncees tuaj yeem kuaj pom los ntawm PAGE electrophoresis; tsis txaus tus qauv; degradation ntawm templates - zam qhov kev taw qhia ntawm impurities thiab rov khov thiab thawing hauv cov qauv npaj; |
| Txc> 38 | Tsawg amplification efficiency;Cov khoom PCR ntev dhau lawm;ntau yam tshuaj tiv thaiv yog degraded |
| Linear amplification nkhaus | Cov kev sojntsuam tuaj yeem ua rau qee qhov degraded los ntawm kev rov ua kom khov-thaw cycles lossis ntev raug rau lub teeb |
| Qhov sib txawv ntawm qhov sib npaug yog qhov tshwj xeeb loj | Cov tshuaj tiv thaiv tsis yog yaj tag lossis cov tshuaj tiv thaiv tsis sib xyaw;Lub thermal da dej ntawm PCR ntsuas yog kis los ntawm cov tshuaj fluorescent |
2.5 Txog kev txheeb xyuas cov ntaub ntawv
Cov ntaub ntawv tsom xam ntawm qPCR tuaj yeem muab faib ua qhov txheeb ze thiab qhov muaj pes tsawg.Piv txwv li, cov hlwb hauv pab pawg kho mob piv nrog cov hlwb hauv pawg tswj,
Ntau npaum li cas lub sij hawm mRNA ntawm X noob hloov, qhov no yog txheeb ze quantification;nyob rau hauv ib tug xov tooj ntawm tes, mRNA ntawm X noob
Muaj pes tsawg daim ntawv muaj, qhov no yog qhov muaj tseeb.Feem ntau qhov peb siv feem ntau hauv chav kuaj yog tus txheeb ze tus qauv.Feem ntau,txoj kev 2-ΔΔctyog siv ntau tshaj plaws hauv kev sim, yog li tsuas yog txoj kev no yuav qhia kom meej ntawm no.
2-ΔΔct txoj kev: Cov txiaj ntsig tau txais yog qhov sib txawv ntawm qhov qhia ntawm lub hom phiaj hauv pawg neeg sim tshuaj txheeb ze rau cov hom phiaj hauv pawg tswj.Nws yuav tsum tau hais tias lub amplification efficiency ntawm ob lub hom phiaj gene thiab lub internal reference gene yog ze rau 100%, thiab tus txheeb ze sib txawv yuav tsum tsis txhob ntau tshaj 5%.
Txoj kev suav yog raws li nram no:
Δct tswj pawg = ct tus nqi ntawm cov hom phiaj hauv pawg tswj hwm - ct tus nqi ntawm cov khoom siv hauv pawg tswj hwm
Δct kev sim pab pawg = ct tus nqi ntawm lub hom phiaj noob hauv pawg sim - ct tus nqi ntawm cov khoom siv hauv cov noob hauv pawg sim
ΔΔct = Δct sim pawg-Δct tswj pawg
Thaum kawg, xam qhov sib txawv ntawm qhov sib txawv hauv qib qhia:
Hloov Fold = 2-ΔΔct (xws li kev ua haujlwm Excel yog POWER)
Yam khoom:
Cell Direct RT-qPCR cov khoom siv
Post lub sij hawm: May-20-2023
