I am trying detect a rna virus in stool samples. To extract the rna, I have been using qiagen viral rna extraction kit. From cultured viruses I had good results but all stool samples came out to be negative. So, to be sure I added pure virus into a stool sample and extracted rna and did rt-pcr (qiagen one step rt pcr kit) but there are still no bands on the gel.
Hi Yavuz,
This sounds like inhibition - something in the stool samples in preventing amplification of the viral RNA.
I recommend trying the following:
1) Use a different extraction kit. The viral RNA extraction kit is designed for 'clean' samples. Perhaps use the Qiagen RNeasy kit or a kit specifically designed for stool samples
2) Dilute the stool samples 1:10 and 1:100 before extraction. Inhibitors tend to be diluted out before viral RNA is diluted out past the limit of detection for the PCR assay. Remember to include these dilution steps if quantifying the RNA
3) Dilute the extracted samples 1:10 and 1:100 before PCR
All the best
Barry
Hi Barrry,
Thanks for the advice but, if I diluted fecal sample, I would also dilute the virus hence, RNA template amount, right?
Hi,
you need to check if there are inhibitors in your RNA, try with your stool RNA in diferents dilutions +/- your viral RNA in differents dilutons too. Also you can try with a total RNA kit or a soil kit: RNeasy PowerSoil Total RNA Kit, because that kind of kits (viral) are used in cell free body fluid.
good luck
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