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RT-PCR


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#1 friend26

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Posted 19 March 2010 - 12:36 PM

I am really new to RT-PCR.
My boss has not much info on this, but wants me to do this.

So here is a short explaination of my work. I am carrying out a dose response test on cancer cells to see the decrease in mRNA levels of 2 genes, with the increase in the concetration of the drug.

Now here is what I do, grow cells, tx them with drug, at 24 hrs, isolate RNA, make cDNA, and then do real time PCR to quantitate.
I use Actin as a standard gene.
I am really clueless, as to what has to be calcualted. I get Cp values which are in the range of 20 to 30 . But what does that convey ?
Should the Cp values decrease as the drug conc. increase.
Why and what does the Actin gene do, when I want to compare it with my other 2 genes.

I am in a total mess here, no one is here to help. :P
Please any advice, tips on how to do and analyse the Rt_PCr will be helpful

Thanks in advance for you responses.

#2 pcrman

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Posted 19 March 2010 - 06:15 PM

Hi,

The purpose of including actin gene is control for potential unequal RNA loading during RT reaction. the Ct values from amplifying the gene of your interest have to be normalized to the actin Ct value from the same sample.

If you expect your gene expression increase after treatment, yet, you will see a smaller Ct than untreated samples.

#3 friend26

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Posted 22 March 2010 - 07:22 AM

Hi,

The purpose of including actin gene is control for potential unequal RNA loading during RT reaction. the Ct values from amplifying the gene of your interest have to be normalized to the actin Ct value from the same sample.

If you expect your gene expression increase after treatment, yet, you will see a smaller Ct than untreated samples.

But how do I do that then. By normalizing to the Cp value to the actin gene, how do I do that. And what does that tell me. So does that mean, with increase in my drug conc, there should be decrease in the Gene expression..which means decrease in the cp value.
So how does that relate to Actin gene, what should I expect for the Cp values in Actin, with increase in my drug conc.
Also smaller the Cp value, more the gene expression, this is how one interprets the result and vice versa ???

Thanks,




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