-RT works better than +RT? - (Nov/17/2008 )
QUOTE
In some condition, -RT can give band, esp on those intronless gene. There is a paper that reported on this and I myself experienced tghe same condition.
Journal of Biochemistry and Molecular Biology, Vol. 35, No. 2, March 2002, pp. 248-250 A Simple Method for Elimination of False Positive Results in RT-PCR
Fátima Martel*, Dirk Gründemann and Edgar Schömig
Journal of Biochemistry and Molecular Biology, Vol. 35, No. 2, March 2002, pp. 248-250 A Simple Method for Elimination of False Positive Results in RT-PCR
Fátima Martel*, Dirk Gründemann and Edgar Schömig
This paper has some technical flaws.
For one, the primer-dimer bands in the gel pic is very clear, almost comparable to the 500bp ladder marker concentration. And for a 10ul load on gel the intensity of the PCR products are not very appreciable. Also it seems that a lot of Taq Pol than normal situations is used. There are also some unspecific bands that were not explained by the authors.
SInce DNase efficiency is reduced in the presence of EDTA, the original RNA might have been diluted in TE buffer, not water. (Thanks Penguin I didn't know that)
DNase treatment also does not guarantee total destruction of your unwanted DNA. Maybe there is more DNA than is originally thought, and the DNase amount that was added is not enough. And the authors did not do a 2nd DNase treatment after -RT to conclusively show absence of DNA in the RT samples.
The limitation of agarose gel imaging also lends to the possibility that there is some DNA left after the RNase treatment (which also acted as a dilution, since only a fraction of the original -RT was taken for RNase treatment). The 2nd Rnase treatment also acted to further dilute the sample. So the sample might have had a very very faint band which cannot be seen by agarose gel and EtBr viewing.
The authors treated their -RT further with RNase. But the -RT reaction also contained RNase inhibitor. Simply exclude RNase inhibitor from the -RT reaction so that the we're sure the RNase inhibitor doesn't inhibit the RNase in any way....
Heck they should just directly use the isolated RNA treated with DNase in PCR and see what's the result.
I'm not saying Taq can't act as a RT enzyme. Maybe it can, but only in very, very specific conditions. If yours is one of those, lucky you. But for others, I would still say Dnase digest a second time and see what you get.
QUOTE
I think it's something else. I didn't see any products in NTC.
When I compared the DNase treated and untreated samples, the products in -RT always appeared before +RT. And after run a gel, I saw the products is around 200bp but is not the size I expected (250). From the gel I also saw the primer dimer which is less than 100bp. But Blast show the primer is specific. sad.gif So I really don't know what's going on in the pcr.
When I compared the DNase treated and untreated samples, the products in -RT always appeared before +RT. And after run a gel, I saw the products is around 200bp but is not the size I expected (250). From the gel I also saw the primer dimer which is less than 100bp. But Blast show the primer is specific. sad.gif So I really don't know what's going on in the pcr.
Your NTC might be clear, but are you sure your RT components (dNTPs, water, etc) are totally Dnase free? Perhaps try RT again with another set. How are the primer designs? maybe your primers have a strong GC clamp on the 3' end that can maybe bind to parts of RNA and amplify from there. Which products are not the expected size? The -RT or the +RT?
QUOTE
i am also facing same kind of problem but here after giving dnase treatment also no rt is rising and that too after 3-4 cycles.i have increaded the amount of dnase used for treatment i.e. 10 U per 10 microlitre of rxn.
The fact that the Cts go up that early already means there there's something wrong. There is saturation of signal. What Cts do your +RT go up?
Molecular biology can be a pain the the a** sometimes with so many things you can't see.
Chris
-chrisbelle-