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RT-PCR problem


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#1 chn09

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Posted 26 March 2010 - 04:06 AM

Hello everyone
I am a Semi Quantitative RT-PCR method to find the expression levels of three target genes. I am using the Qiagen One Step RT-PCR kit for this. I am using the GAPDH gene as the internal control for the semiquantitative RT-PCR. I find that in one of the experiment the GAPDH is expressed at a high level, and in another three, it is expressed at a lower level. Why is it that an internal control such as GAPDH fluctuate? I have used less than i microgram of RNA i.e 5 ng for the semiquantitative RT-PCR. And also i did the RT in triplicate. still the problem appears. Pls help

#2 ivanbio

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Posted 26 March 2010 - 06:46 AM

While GAPDH has been used for a very long time as an "internal control", the fact of the matter is that this gene does fluctuate in expression in some tissues/conditions. Your experimental conditions seem to be one of those where GAPDH changes in gene expression. Your best bet is to try other internal controls to find one that really works as an internal control (i.e. does not fluctuate in gene expression under your experimental conditions).

Good luck

Ivan
Carlsbad, CA




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