RNA Gel Results showing 28S:18S ratios. (Image kindly provided by tebu … · kindly provided by...
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Figure 1. RNA Gel Results showing 28S:18S ratios. (Image kindly provided by tebu‐bio laboratory's RNA extraction and Genomic Profiling Service division). Figure 2. ImageJ quantification of 28S:18S ratio. First measure the background (left), then measure the 28S band (middle), then measure the 18S band (right). Subtract the background from the 28S and 18S values and calculate the ratio.
Transcript of RNA Gel Results showing 28S:18S ratios. (Image kindly provided by tebu … · kindly provided by...
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.
