Page 15 - Demo

263ANALESRANFwwwanalesranfcomMaternalfructoseintakemodulatescholesterolmetabolisminresponsetoaWesterndietintheoffspringPalomaLópezLaizElenaFausteetalAnRealAcadFarmVol88nº3(2022)·pp257-275–BaselinetheinitialamountofdsDNAisminimalandthefluorescenceintensityistoolowtobedetected–Exponentialwhenthedetectionthresholdhasbeenoverpassed theintensitychangesexponentially. ThecycleatwhichthefluorescenceisdetectedandstartsraisingexponentiallyiscalledCrossingPoint(Cp) ThisparameterisinverselyproportionaltotheinitialquantityofDNAThusthelowertheDNAamountthehigherthenumberofcyclesrequiredtogetthesignaldetectedresultinginahigherCpvalue–LinearthereactionratedecreasesbecausetherearenotenoughDNApolymerasemolecules togrowall theDNA strandsalreadyproduced–PlateautheDNApolymeraseissaturatedsothesignalismaintainedconstantresultinginaplateauregionTheinitialamountofcDNAcanbequantifiedusingacalibrationline Tocreateit serial1/10dilutionsofa standard samplewithaknownDNAconcentrationwerepreparedandtheirCpobtained Then thecDNAconcentrationof thesamplesisobtainedbytheinterpolationoftheirCpvalueinthecalibrationline(InvitrogenTMUSA)AfterthisprocedurebothanormalPCRandanagarosegelelectrophoresis(Figure6) wereperformed toensurethat thecDNAobtained was functional ThisPCR wasdone withprimersthatbindtoahousekeepinggenesequencetheribosomalproteinS29(RPS29)gene AsshowninFigure6allcDNAsamplescorrectlyamplified theproductofRPS29geneconfirming the functionalityofthecDNAthathadbeenobtained33cReal-TimePolymeraseChainReaction(Real-TimePCR)33caBasicprinciplesofthemethodThereal-timePCRisatechniquethatallowsthequantificationof theDNApresentina sampledue to thedetectionof fluorescentparticlesthatbindspecificallytothedoublestrandedDNA(dsDNA)(Figure7)ThemeasurementoffluorescentintensityisperformedaftereachcycleofPCRbecauseasitis thepointat whichmoredsDNAispresentthesignalwillbegreater.ThefluorophoreusedwasSYBRGreenIwhichbindspreferentiallytodsDNATherateat whichDNAisgeneratedisnotlinearand therearefourstagesinitsproductionaccordingtotheintensityoffluorescencedetected(48)Figure6AgarosegelelectrophoresisofRT-PCRFigure7 Schemeof themechanismof theReal-TimePCR The fluorophore(SYBRGreenI)isonlyable toemit fluorescencein theexcited stateie whenbound todoublestrandedDNA(dsDNA)CreatedwithBioRender.com