DNase treatment of RNA prior to RT-PCR - (Nov/08/2006 )
Even though I have done many experiments successfully I would like to know the proper method of DNase treatment of RNA. Like how would you decide how many units you need (NEB DNase says 1 U can digest 1ug DNA). And also the duration sufficient for complete reaction. Because I see many people having problem with bands in No RT control. Thanks.
usually I use Turbo DNA-free from Ambion, this works fine.
It depends of course how you actually isolate your RNA; Qiagen columns offer DNA digestion straight on the column.
Another option is to design your primers exon-spanning.
Regarding this Ambion stuff I stick to the routine DNase treatment ('rigorous' is another option); but until now I had no big trouble with signals in no RT-control.
Most of the time I use TRI-reagent (Sigma) or Trizol for RNA isolation. I do a PCR using RNA as a tempate. If I see a band there I go for DNase treatment.(most of the time which is necessary). When I have very few cells then I use Qiagen RNeasy columns and do on membrane DNase treatment. Even in this case smetimes I see bands in NoRT control.
i use too the Turbo DNAse from ambion
You could use Shrimp nuclease (USB, GE, ABgene). Then you would need far less than 1 U, and you would not need to worry about getting rid of it, - it's very quickly inactivated by heat.
Since it only digests doublestranded DNA, you could add it together with the RT, saving even more time.