I am working with tumor, precancerous and normal tissue from hamster cheekpouches.
I am not able to detect the expression of GADPH, b-Actin, Rpl13a and Tubb housekeeping genes with qPCR in normal and precancerous tissues. I have tried treating with DNase I in case of genomic DNA contamination but still nothing. I have no problem in detecting these genes in tumors from the same individual. Any idea what could be happening?
Thanks!
Carla
First, a simple question, did you check the RNA quantity and quality of all samples and used roughly the same amount of RNA as input for reverse transcription?
Sabine Strehl
Hello !
I used the same amount in tumor and normal tissue samples, 2 ug each (Following Promega MMLV protocol). The amount of RNA went from 0.5 to 4 ug/ul and the absorbance ratio 280/260 was >1.9. I tried RT with DNasa I to eliminate probable contamination with genomic DNA but also nothing.
Vipul Batra
Hello!!
I tried 4 different housekeeping genes, GADPH, b-Actin, Rpl13a and Tubb. Actually I got them from a paper entitled "Validation of housekkeping genes in hamster" which is my model. It is really strange because I had no problems with these genes un tumor tissues with any genes. I choose Rpl13a just because showed to be the most stable.
I meant non-reference genes. Are they problematic as well? If yes, you gotta start over from evaluating the quality of isolated RNA.
Ah ok. Right, no, I can´t detect the gene of interest either. I know at this point that there is a problem with the RNA but I don´t know how to solve it. I have no problem with tumor RNA.
How did you evaluate the quality of isolated RNA? Afterwards, may be we can go through the protocol that you follow to figure out what's wrong.
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