RNA extraction, RT-PCR problems
jhan21 at gmail.com
Mon Apr 24 17:01:53 EST 2006
Hoping that someone out there might have had a similar problem or have some
insight on this...
We extract total RNA from mouse tail skin using a combined Trizol/Qiagen
miniprep protocol and use a 2 step real-time RT-PCR protocol to examine gene
The isolated RNA is reverse transcribed into cDNA (using random hexamers and
other reagents from Applied Biosystems) and run in TaqMan RT-PCR. Now for
For any given batch of samples, we always experience a few samples that give
no amplification product in the RT-PCR despite equal handling of all samples
during RNA isolation, reverse transcription, and RT-PCR. Initially, we
assumed error during the RT-PCR procedure and ran another plate with all
those samples, but the same samples that were bad the first time yielded no
product again. Then, we wondered if certain of the cDNA samples were bad
because of error during the reverse transcription procedure, so we re-did
the RT with all samples and ran another RT-PCR with the newly made cDNA.
Again, the same samples yielded no product. Thus, it must mean that for
these samples, the RNA was bad in the first place...
However, all of our isolated RNA samples have good optical density and good
280/260 ratio (~2.0). We've also tested the integrity of the RNA by gel
and Bioanalyzer, and both methods indicate the RNA is present and intact.
But for some reason, certain samples will give us good RT-PCR amplification
while others will yield no product.
This has happened to us pretty consistently for a while and we've tried
troubleshooting to no avail. There should be no reason why a subset of
samples would be unable to yield PCR product, especially since the RNA
appears equally good across all samples.
Any thoughts or suggestions of things to try would be greatly appreciated.
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