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Data Analysis for Real-time PCR - (Mar/11/2010 )


I use the STEPONEPLUS Real-time PCR instrument from Applied Biosystem. As far as I understand and have experienced, if your gene is expressed at very low levels, the Ct values are generally higher. (above 35). But if the gene is not expressed or below detection levels by the instrument, the Ct values show up as UNDETERMINED. I run my samples in triplicate and for such low expressing genes, I get results with very high Ct values or undetermined. How do you report this scientifically, since there is not really a method to quantify, if the signals are beyond the instruments detection limit.




why dont you set up -RT controls for your gene of interest also. even if your Ct values are high in your sample, the -RT controls should give you the value as undetermined. Also, if your +RT is showing undetermined value, you can increase the number of cycles and compare the curves of your +RT and -RT samples.


Report as Ct > 35 (or whatever).