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Strange control contamination in cDNA synthesis

Taqman miRNA assay

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2 replies to this topic

#1 GoldEpi



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Posted 07 June 2013 - 03:49 AM

Hello everybody,

With one miRNA and its control (U6), I get two cDNA control contaminations: one without RTase and one without RNA template. The curves are normal and the Ct values are even better than with normal samples! The opposite controls are fine, so the only difference i.e. the primer (present in every well) cannot explain the case alone. qPCR master mix controls are fine, too.

Everything has been double-checked. Does anyone have some ideas? Thanks in advance.

Edited by GoldEpi, 07 June 2013 - 05:14 AM.

#2 pcrman



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Posted 16 June 2013 - 08:40 PM

There could be multiple sources of contamination: primers, components of RT reaction, etc. Did you DNase your RNA?

#3 GoldEpi



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Posted 29 June 2013 - 06:30 AM

Thanks for answering. I did not DNase my RNA but the sample is so limited that it is not even possible I think... It is just so weird that contamination can be combination-specific with such a big difference...

And if I was not clear enough, the contaminations are:
U6 RT primers + no RTase + RNA + qPCR master mix
miRNA RT primers + RTase + no RNA + qPCR master mix

Edited by GoldEpi, 29 June 2013 - 06:34 AM.

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