When is the right time to adjust RNA concentrations for Rt-PCR? - (Aug/08/2008 )
I run several sets of RT-PCRs everyday and usually, I am comparing the effect of different conditions on the expression of the same gene.
For having an accurate comparison, I set the RNA concentrations of my samples to the same (I use Biorad spectrophotometer). But still, I don't know when is the right time to do it. I do it after RNA extraction and before DNAse treatment (Promega). Is it proper? If it's possible to do it after DNase treatment, how could I get rid of enzyme (DNAse) inside the tube?
(I run a housekeeper gene along with mt samples anyway, but still I do need to set my samples to the same!)
if you measure it before DNase treatment the measurement can be affected by DNA which also absorbs at 260nm.
I would say a Phenol chloroform extraction or LiCl precipitation would do the job.
Whenever I am working with RNA I do phenol/chloroform extraction then check the concentration. I adjust my concentrations right before I run RT. I have never treated with DNAse for anything I've done with RNA thus far.