Determine the RNA concentraiton for linear PCR - (Mar/11/2005 )
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!
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.