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cDNA and RT PCR when CT of house keeping gene is ranging


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#1 Lilip

Lilip

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Posted 09 June 2011 - 04:52 PM

Hey, I am new here -just for creating a topic- Ive been reading so many topics for months and lot of them were very helpful!

I run a PCR for GAPDH gene using cDNA prepared with 1microg of RNA in a total volume of 20microl. The result was CT varying to much between them.
The most obvious reason i see is I made a mistake when pipetting RNA to performe cDNA. But I could say i have some (!) experience on lab.
Other reasons just came up with:

1- Is it possible that the amount per microl had ranged too much on Nanodrop and than i got different amounts instead of 1microl to prepare cDNA?

2- I performed quality gel of my samples and was Ok. But there were bands thicker than others. I was wondering if is it possible to have a high quality RNA but, for example, with 20% rna degraded. So I picked 1microg when i was preparing cDna and actually i had 800nanog.

I would appreciate your answer.
PS. Sorry for my english

Edited by Lilip, 09 June 2011 - 04:57 PM.





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