Hi!
In our lab, we are currently having a lot of trouble with extracting RNA from rat peritoneal mast cells. Mast cells were isolated from rat peritoneum and a method of Chomczynski was used to extract the RNA. We have obtained yields of 0.8 ug/uL RNA (from 0.5x106mast cells); A260/280 ~ 1,99. But in qRT-PCR we obtained unsatisfactory results.
Thanks a lot.
Elzbieta
- inhibitors in RNA solution
- RNA is degraded: spectrophotometer does not show this
- how much cDNA was used for qPCR? Maybe matrix inhibition???
- Try any IPC in your qPCR
- use 1 to 100 ng cDNA per 20 µL reaction for PCR.
-http://www.jimmunol.org/content/178/10/6465#sec-21
Maybe it would be helpful. Best Regards,
Akif
Dear Elżbieta Kozłowska,
Can you describe your problems with PCR in more detailes? It is likely that the problems are not related to RNA isolation, but to subsequent stages.
Best regards,
Maxim
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