I am currently engaged in a project, where I want to quantify mRNA levels of several genes (some of them very low abundance) via RT-qPCR. None of the genes includes introns, so -RT (no Reverse Transcriptase) controls are essential for confirmation of validity of my experiments.
As far as I saw from reading prevoius threads, I am by far not the only person having the problem of eliminating gDNA contamination by means of DNase treatment. I extract RNA with QIAGENs RNeasy, usually digest with 3U of RQ1 DNase (Promega) in provided buffer for 1h and cleanup with RNeasy before RT with Invitrogens Superscript. For RT I used either random hexamers or GSPs.
I found previous entries very helpful and it seems to be clear (as posted before) that a 100% elimination of gDNA contamination by DNase treatment without seriously compromising RNA quality or reducing sensitivity of qPCR (e.g. less cycles) is not possible.
Because of that I was looking for alternative methods, circumventing DNase treatment. I found two articles, which appeared very interesting :
RNA template-specific polymerase chain reaction (RS-PCR): a novel strategy to reduce dramatically false positives.
Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.
So far I didn´t get access to full text (hopefully this will change soon), but the general approach seems appealing to me. As far as I understand they use a GSP for first-strand synthesis including a 5´-tag-sequence, that is not present in the gDNA. This tag-sequence, of course, doesn´t anneal to the RNA, when rverse transcribed, but will appear in the resulting cDNA. The tag-sequence is then used as reverse primer in the subsequent PCR reaction, giving rise to a product only for amplification of cDNA.
BUT even though this doesn´t seem to be a very new idea (1990 or earlier) I haven´t seen many publications apllying this technique. Also (excuse me if I´m wrong) I didn´t find a topic/discussion of that approach in this forum, even though a lot of people appear to be struggling with gDNA problems. Since you (Suzanne and others) seem to have a great deal of experinece in the field, I would love to hear your oppinion!
There is also a freely available program for designing proper tags for this method (in perl):
Anyone who tried that, I would love to hear about results!
Many thanks in advance for comments and experiences with this method!