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Determine the RNA concentraiton for linear PCR


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#1 Daniel5306

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Posted 11 March 2005 - 04:53 PM

Hi, there,

I am trying linear PCR to determine the expression of 6 genes in different condition. I use GADPH as a control, to normalize the concentration of gene in different sample. I repeat the experiment for two times. The problem what I met was that even the product of GADPH changed significantly among different sample. Using the same cDNA, I repeated two time, the result repeated very good, which indicated the problem was not from PCR, it should be from RT reaction. A possible reason is that the mRNA, used to reverse transcribe the cDNA, is not in the same concentration. What I did was, after being resuspended in water for 10mins, the concentration of RNA was measured by photometer at 260nm, then added the same amount of RNA in different sample. I don't know how to improve it, and hope to hear some advice from experter. Thank you in advance!

Edited by Daniel5306, 11 March 2005 - 04:55 PM.


#2 pcrman

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Posted 11 March 2005 - 06:05 PM

Hi Daniel,

The inconsistence may be caused by many variables such as the initial quantitation of RNA with a spectrometer. You may get ten different OD readings if you repeat ten times especially if samples are not mixed well. Other vaiables include RT and PCR efficiency, human error. I have found one-step RT-PCR works better than two-step PCR because the former reduces some error prone steps. Also check the lamps of your gel documentation system to make sure they light equally.




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