Download - RNA Gel Results showing 28S:18S ratios. (Image kindly provided by tebu … · kindly provided by tebu‐bio laboratory's RNA extraction and Genomic Profiling Service division). Figure

Figure1.RNAGelResultsshowing28S:18Sratios.(Imagekindlyprovidedbytebu‐biolaboratory'sRNAextractionandGenomicProfilingServicedivision).

Figure2.ImageJquantificationof28S:18Sratio.Firstmeasurethebackground(left),thenmeasurethe28Sband(middle),thenmeasurethe18Sband(right).Subtractthebackgroundfromthe28Sand18Svaluesandcalculatetheratio.

Figure3.ImageJProfilePlotassessingRNAquality.UsingthisRNAgelimagefoundontheinternet,theImageJ(Analyze‐ProfilePlotFunction)wasusedtodrawahistogram,showingalow28S:18Sratioandsignificantsmearingbelowthe18Sband,indicatingpoorRNAquality.

Figure4.SampleAgilentBioanalyzerResults.Usingamicrofluidicsapproach,theAgilentBioanalyzerdeterminestherelativeabundancesof28Sand18SRNAaswellasthedegreeofdegradation.ThesoftwarecreatesdigitalplotsthatresembletraditionalRNAgels(left)andcalculatestheRNAIntegrityNumber(RIN)basedontheplottedhistogram(right).

Figure5.Meltingcurves.Acceptablemeltingcurvesarethoseshowingameltingcurve(blue)distinctfromthemeltingcurveyougetwiththenegativecontrol(yellow).Agoodprimerpairwillgiveaspecificpeak(left)whileunacceptableprimerpairswillyieldproductswithlesspuremeltingcurves(middleandright).

Figure6.AmplificationCurves.AcceptableamplificationcurvesareC‐shaped(left).cDNAthatistooconcentratedmayyieldanS‐shapedcurve(right),howeverfurtherdilutionofthesampleincreaseCtvaluestothemeasurablerangeof20‐30.

Figure7.qPCRStandardCurves.AcceptablestandardcurveswillhaveanR2near1.0andanEfficiencyof100%orless.