dna contamination after turbo free dna
Posted 22 May 2011 - 11:21 PM
I had variability in my qRT-PCR results and my supervisor recommended me to treat my RNA samples with DNas1 because in our lab we used to isolate RNA without DNase treatment so we bought Turbo DNA free from ambion but unfortunately it didnít work. I fellow the protocol but i got very diluted RNA samples and after I run qRT-PCR I got more variability even in house keeping gene. Anyone use turbo DNA free, please tell me exactly how its work and how much RNA sample you use and what is the reaction final volume.
Thanks in advance
Posted 23 May 2011 - 04:22 AM
Your RNA may degrade during the procedure (do you use RNase Free water?) or there may be too much DNA. To check whether your problems are due to DNA contamination, run an RT- reaction (substitute reverse transcriptase with water in RT reaction) and see how much DNA you have in your samples.
And of course after DNAse treatment you have to measure RNA concentration and add the same amount to each RT reaction, you can't use measurements done before that.
Just a few thoughts..
I never trust anything that can't be doubted.
Posted 23 May 2011 - 06:11 PM
really appreciate your help...