Online life science pharma marketplace and platform for products and services.


Current location: Home » Ask » Molecular biology » Content

not reproducible : reverse - transcription PCR

Pending answer Credit reward to answer the question: 0 -
More information:
Could anyone please help me with a problem in my semi-quatitative RT-RCR? I attempt to compare the transcript level of a gene in two samplers. I got very good result last week---- sampler A has dramatically higher expression of that gene than sampler B. B-actin was used as loading control and 1:4:16 serial dilutions of both B-action and the interested gene was performed.

But when i tried to reproduce this result this week, everything goes wierd. The dramatic difference between the two samplers disappeared. And the B-actin control looks strange-- not following 1:4:16 pattern and not the same between two samplers. I have been trying repeating the experiments for a whole week but the results were always like that. I am wondering whether anyone else has similiar experience in non-reproducibility of semi-quatitative RT-PCR? Any suggestion for me to fix the problem?

I think the number of cycles in my PCR program might be one problem. I was using 40 cycles because my mRNA was extracted from only 1000 cells. I could reduce it to 35 cycles but probably cannot go any further.
total 540 hits     Asked by: Anonymouse I want to answer  

[ AskSearch ]  [ ]  [ Tell a friend ]  [ Print ]  [ Close window ]  [ Return to top ]