Dear Iris Steitz ,

We are using DNaseI from Promega to prepare your samples for reverse transcription and subsequent real-time PCR for the last 15 years. Usually we are preforming the DNase step in the RT-buffer.

I have designed most (but not all primers) that they either span two Exons or that the Intron in the genomic sequence is so big that a product can not be formed.

Nonetheless, we still have some primers which will also give a signal with gDNA. Therefore, we are using a RNA control where only the digestion was done but no reverse transcription. These samples also gave some signals in the past but only at high Cts.

And when now a RT sample would come at a Ct 23 and your RNA control is coming at a Ct of 33 there is not really a problem, since that would mean, that the gDNA in your RT sample would only make for 1024th of the samples signal and a measurement with a 1/1024 systematic error should still do fine.

Kind regards

Soenke

Ensuring Complete Removal of Genomic DNA: Optimize DNase Concentration and Incubation Time, Use of gDNA Eliminator Columns, Check for Contamination, Perform Double DNase Treatment

Reliable Sources for RNase-Free DNase:

Thermo Fisher Scientific, QIAGEN RNase-Free DNase Set, New England Biolabs (NEB)

I use TURBO DNase from Invitrogen to remove DNA from RNA samples, it is RNase-free. But if you are getting lots of gDNA in your RNA samples then you should focus on improving your RNA preps. Either switch to a different RNA extraction kit, or use phenol chloroform extraction. With phenol chloroform extractions you should be able to get higher yields with no contaminating gDNA.