Internal control for RT-PCR analysis - The expression of internal control (Apr/24/2008 )

Hi all,

I'm trying to analyze the mRNA distribution in various tissues such as liver, muscle, brain, etc.

The problem I'm facing now is the expressions of internal controls (beta-actin, GAPDH) vary among the tissues even though i apply the same total RNA amount in all samples during RT-PCR. I did optimize the PCR cycle so that the amplification is in the exponential phase.

Anything that I did wrong or these two genes are not good as internal control? My colleague told me that we can't get even expression for internal control, is him right? I also come across with some papers show even expression in different type of tissues for internal control such as beta-actin.

I hope someone can shed light on this topic. Thanks and have a nice day.

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Your colleague is correct.

There is NO such gene that consistently express among different tissue, individual subject (animal or human) and verious treatment.
If compare among tissues, the 2 you choose are NOT going to work.
The best one for internal control among tissue is rRNA, but it is too much of them for PCR to work.

We have try 36B4, I cannot recall its full name sorry ; it is a mRNA ribosomeal protein and I have used it for more than half of my PCR.
I do internal control selection (4 gene first HPRT, 36B4, beta-actin and PPIA) from at least 1/4 of my samples before I use all samples.
I usually get relatively good result from these 4; if these 4 are not good enough then I try some others.

Currently there are software facilitate normalization processing from mulit-internal control PCR.
You may consider that also

Hope this help

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Hi wuxx0153,

Thanks for the sharing.

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