-RT works better than +RT? - (Nov/17/2008 )

Hi bodies,

When I do q-PCR I met a weird problems. In my minus RT control, there are products appeared and more surprisingly, it works even better than +RT. And in both case, the Ct value is around 25-27, which may exclude the possibility of reverse transcriptase activity of Taq DNA polymorase. I was so confused and could anybody help me to figure out possible explanation and solution?

Thanks in advance.
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Eric

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Do you treat the RNA with Dnase before doing the cDNA??? If you use a column base rna extraction sometimes it left trace of DNA.

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Yes I did perform DNase treatment and compared with untreated one. After DNase treatment, -RT did work better than those were not treated. However, in some genes I tested, -RT worked better than +RT. I doubt the interfere of RNA left in the -RT, but I couldn't explain the results.

Thanks for reply.

Eric

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the only reason why your -RT control gives you such good Ct is that you have contamination that can be amplified by your primers. how is the NTC control? I would say you still have DNa contamination. do DNase treatment again. or there something seriously wrong with your whole protocol.

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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. So I really don't know what's going on in the pcr.

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PCR needs a DNA template. If you're getting a product in your -RT control, you have DNA contamination. It's just that simple.

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hi,

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.

I dont know what to do?
Please help me.

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Hi there

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

Hope this help

Cheers

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Hi there

My personal experience told me that in usual cases the DNA contamination in RNA sample is not that bad. Since you are isolating RNA, it is a bit impossible for you to contaminate it with 50% DNA (ie in a solution 50% is RNA, 50% is DNA). I have tried to run my DNase treatment protocol on a pure DNA sample. If such a great amount of DNA (pure DNA sample comapring to the minute contamination of DNA in RNA sample) can be degrade by the DNase treatment, thus increasing the DNase unit won't help much in your case.

Hope thsi help, and do correct me if I am wrong.

Cheers

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This may sound obvious but are you sure the DNAse enzyme is functioning? DNAses need Mg2+ to work efficiently so if you don't add in MgCl2 (usually included in the DNAse buffer) or if you have EDTA in your reaction then not all of your DNA will be cleaved.

P

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