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Leaky RT-PCR


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4 replies to this topic

#1 marek82313

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Posted 20 June 2009 - 06:08 AM

Hi,
I've a question about an oddity that appens when I perform PCR experiments with some cDNA.
I've synthesized 4 cDNAs from the RNAs from 5 different cell lines using random hexamers, now I'm performing some PCR experiments to verify the presence of some RNAs I'm studing. The number of loci I'm studing is 6, for each one I've 2 primer pair, one of which is a nested primer pair. 2 of the loci are even positive, each time I perform a PCR I obtain a fragment of the expected size, but the results for the other four are not always the same!
When I performed the PCR for the first time I obtained a results, the second time I did the experiment using the same condition the result was different! Some of the samples that amplified the first time didn't amplify during the second experiment, other samples that didn't amplify in the first experiment amplified in the second.
I tried performing the experiments using the nested primers too, and sometimes the results obtained were different from the ones obtained using the external primer pair.

Did someone experiences this oddity?

Is is possible the RNA I'm looking for is a low copy one so the number of the First Strand cDNA molecules is so much low that sometimes the PCR reaction fails?

#2 pcrman

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Posted 21 June 2009 - 12:45 PM

What are the size of your amplicons and why do you use nested primers for RT-PCR? Did you including RT- and Water controls?

#3 marek82313

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Posted 21 June 2009 - 01:16 PM

The size of the amplicon ranges from 100-300 bp when the external primers are used, and about 90-100 bp when the nested primers are used. I designed the nested pairs because I'll need to perform some 5'RACE experiments, and I begun using them in PCR experiments when I started obtaining these odd results, thinking that someone of the external primers could have some sort of problem with the annealing . I ever include RT- and water controls, they are clean, I've never seen DNA contamination in these controls.

#4 pcrman

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Posted 21 June 2009 - 08:17 PM

The loci you are amplifying seem to have low expression. Beware that any DNA contamination may give false results unless you have done DNase treatment. You said your primers are for 5' RACE, so they are at the 5' end of the genes. So also beware that some genes have uncertain 5' end.

#5 marek82313

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Posted 22 June 2009 - 12:38 AM

Ok, thanks!




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