trouble with DNase treatment of RNA sample - (Feb/04/2008 )
I isolated microRNA fraction of total RNA from mouse brain tissue (~18ng/µl). I tried to do DNase treatment but always got bands on the noRT PCR controls (using 5S or U6 primers). How do you guys control for successfull DNase digestion? how many PCR cycles? which DNAse you prefer to use?
Thanks a lot!
If you use 5'-tailed RT primer, you don't need to remove the DNA. Check out the details here: Chen, et al. Real-time quantification of microRNAs by stem–loop RT–PCR, Nucleic Acids Research 2005 33(20):e179 (FREE Full Text and Supplementary Data).
You don't need to remove DNA if you use PolyA tailing based RT-PCR for miRNA, since there's only one gene specific primer in PCR reaction...
Thanks a lot guys!
I have the Taqman probes, but didnot try them yet. what I used before was SYBR green, and I get funky results (multiple bands etc); also it seems to be difficult to get rid of DNA contamination (I guess since the resulting chunks should be smaller than the amplified fragment).
thanks again, I'll try the Taqman probes.