DNase treatment of RNA - (Nov/02/2006 )
Hello- is it necessary to add DNase to RNA after it has been extracted w/ Trizol and ethanol precipitated? If so, whats the best protocol...i'm assuming 1uL of DNase and a 10 min incubation at 37 deg. any suggestions?
It's necessary to avoid false-positives provoked by possible DNA in your sample. I used to do DNAse treatment with 1ul DNase (or 10 units) for 30 min at 37ºC.
I was told when doing Dnase treatment to be aware of the buffer the dnase is supplied in. Some buffers may cause complications in down stream applications. If using the qiagen kit, their dnase treatment is nice in regards that it is washed away after incubation on the column. I use the Ambion Ribo pure kit which is a combination of trizol and column based purification (skeletal muscle as tissue), and I don't do a dnase step. I always run a reverse transcription run without reverse transcriptase and use that as a neg control in my real time pcr and I have never had signals from that. (knock on wood )