分段書寫3mm d pcr顱底軟骨細(xì)胞分離和培養(yǎng)

WesternBlot分析COL2A1RNATotalRNAwasextractedfromCBSCsusingTrizol(TaKaRa,Japan)accordingtothemanufacturer’sspeci?cations.TheyieldofRNAwasdeterminedusingaNanoDrop2000spectrophotometer(ThermoScientific,USA),andtheintegritywasevaluatedusingagarosegelelectrophoresisstainedwithethidiumbromide.?cationwasperformedwithatwo-stepreactionprocess:reversetranscription(RT)andPCR.EachRTreactionconsistedof0.5μgRNA,2μlofPrimerScriptBuffer,0.5μlofoligodT,0.5μlofrandom6mersand0.5μlofPrimerScriptRTEnzymeMixI(TaKaRa,Japan),inatotalvolumeof10μl.ReactionswereperformedinaGeneAmp?PCRSystem9700(AppliedBiosystems,USA)for15minat37℃,followedbyheatinactivationofRTfor5sat85℃.The10μlRTreactionmixwasthendiluted×10innuclease-waterandheldat-20℃.Real-timePCRwasperformedusingLightCycler?480ⅡReal-timePCRInstrument(Roche,Swiss)with10μlPCRreactionmixturethatincluded1μlofcDNA,5μlof2×LightCycler?480SYBRGreenIMaster(Roche,Swiss),0.2μlofforwardprimer,0.2μlofreverseprimerand3.6μlofnuclease-water.Reactionswereincubatedina384-wellopticalte(Roche,Swiss)at95℃for10min,followedby40cyclesof95℃for10s,60℃for30s.Eachsamplewasrunintriplicateforysis.AttheendofthePCRcycles,meltingcurveysiswasperformedtovalidatethespeci?cgenerationoftheexpectedPCRproduct.TheprimersequencesweredesignedinthelaboratoryandsynthesizedbyGenerayBiotech(Generay,PRC)basedonthemRNAsequencesobtainedfromtheNCBIdatabase(Table1)TheexpressionlevelsofmRNAswerenormalizedtoGAPDHandwerecalculatedusingthe2-ΔΔCtmethod(LivakandSittgen,2001).表3各擴(kuò)增使用的引物序列、循環(huán)數(shù)和產(chǎn)物大Table3NucleotidesequencesoftheprimersandprobesusedforRT-產(chǎn)物大小代及傳代過程中軟骨相關(guān)Col1a1，Col2a1，Col10a1及Sox9的表達(dá)量變化。液，吹打至清亮，轉(zhuǎn)移至EP1mlTrizol200ul15s，常溫靜15min；412000g15min500ul，顛倒混勻，10mi（RNA提取效率℃12000g10min，RNA1ml75%乙醇（DEPC水配制混勻。4℃7500g離心5min，去上清，樣品干燥5-10min，用RNA-水（DEPC水1：1000稀釋）20-50ul30minNanoDrop2000分光光度計(jì)測(cè)定濃OD260/OD280RNA完整性。轉(zhuǎn)錄反應(yīng)完全后，85℃5s終止反應(yīng)。逆轉(zhuǎn)錄完畢后加入90μlNuclease-H2O稀釋至100μl在-20℃冰箱，用于后續(xù)實(shí)驗(yàn)。引物設(shè)計(jì)：引物采用RocheLCPDS2軟件設(shè)計(jì)并由捷瑞生物工程合成，各擴(kuò)增使用的引物序列、循環(huán)數(shù)和產(chǎn)物大小見表3。PCRLightCycler?480SYBRGreenIMasterLightCycler?4802×LightCycler?H2O，3.6μl。PCR程序：95℃10min；95℃10s，60℃30s，40個(gè)循環(huán)。循環(huán)結(jié)束后利用熔解曲線檢測(cè)產(chǎn)物特異性，從60℃緩慢升溫至97℃，每℃5次熒光信號(hào)。相對(duì)表達(dá)量，ΔCt=Ct目的-Ct內(nèi)參，ΔΔCt=ΔCtP1-P4樣品-ΔCtP0樣品，相對(duì)表達(dá)量=2-ΔΔCt。應(yīng)用test單因素方差分析（One-wayysisofvariance，ANOVARNATotalRNAwasextractedfrom(inputthetypeofyoursamples)using(inputthekitusedforRNAextraction)accordingtothemanufacturer’sspeci?cations.TheyieldofRNAwasdeterminedusingaNanoDrop2000spectrophotometer(ThermoScientific,USA),andtheintegritywasevaluatedusingagarosegelelectrophoresisstainedwithethidiumbromide.?cationwasperformedwithatwo-stepreactionprocess:reversetranscription(RT)andPCR.EachRTreactionconsistedof0.5μgRNA,2μlofPrimerScriptBuffer,0.5μlofoligodT,0.5μlofrandom6mersand0.5μlofPrimerScriptRTEnzymeMixI(TaKaRa,Japan),inatotalvolumeof10μl.ReactionswereperformedinaGeneAmp?PCRSystem9700(AppliedBiosystems,USA)for15minat37℃,followedbyheatinactivationofRTfor5sat85℃.The10μlRTreactionmixwasthendiluted×10innuclease-waterandheldat-20℃.Real-timePCRwasperformedusingLightCycler?480ⅡReal-timePCRInstrument(Roche,Swiss)with10μlPCRreactionmixturethatincluded1μlofcDNA,5μlof2×LightCycler?480SYBRGreenIMaster(Roche,Swiss),0.2μlofforwardprimer,0.2μlofreverseprimerand3.6μlofnuclease-water.Reactionswereincubatedina384-wellopticalte(Roche,Swiss)at95℃for10min,followedby40cyclesof95℃for10s,60℃for30s.Eachsamplewasrunintriplicatefor ysis.AttheendofthePCRcycles,meltingcurveysiswasperformedtovalidatethespeci?cgenerationoftheexpectedPCRproduct.TheprimersequencesweredesignedinthelaboratoryandsynthesizedbyGenerayBiotech(Generay,PRC)basedonthemRNAsequencesobtainedfromtheNCBIdatabaseasfollows:(inputyourprimerTheexpressionlevelsofmRNAswerenormalizedto(inputthereferencegene,e.g:GAPDH,ACTB)andwerecalculatedusingthe2-ΔΔCtmethod(LivakandSittgen,TotalRNAwasisolatedusingTRIzolreagent(Invitrogen,Carlsbad,CA)accordingtothemanufacturer’sinstructions.Afterreversetranscriptionreaction,realtimereversetranscription-polymerasechainreaction(PCR)wasperformedbyaRoc