Protocol Online logo
Top : Forum Archives: : Molecular Biology

DNase digestion step necessary for real-time RT-PCR? - (Jan/22/2007 )

I am extracting RNA from microglia using the QIAGEN RNeasy Kit for use in real-time PCR. Should I do a DNase digestion step before reverse transcription?


If your Qiagen RNeasy kit includes the DNase treatment onto the membrane its better to do it always. If you dont want then do a PCR for your RNA as template with a set of primers which can also amplify genomic DNA for atleast 35 or 40cycles. If you see no amplification in No RT control (ie your rna) then no need for DNase treatment. But if you see even a faint band its recommended to do DNase treatment. So I prefer incuding DNase treatment for all RNA samples especially for Real Time PCR.


With RNeasy you will always have gDNA contamination, as opposed to TRIzol reagents etc.. That is just how the kit works, since after cell lysis also gDNA can bind to the column. For RT it is important to get rid of gDNA since it can give wou wrong signals in you expression analysis. It will take you less than 30 min and save you a lot of troubles later.