do you subtract gel base intensity from PCR band intensity? - urgent require (Jun/22/2007 )
Hi, dear colleagues,
I am comparing mRNA expression levels of cardiomyocytes under different culture condition.
PCR products were agarose gel electrophoresed and ethidium bromide stained. The resulting band intensities were measured by means of the public NIH image processing program (Image J, http://rsb.info.nih.gov/ij/index.html). For subsequent analysis of each target mRNA, the relative expression quantity was evaluated by the ratios of band intensity to GAPDH and β-actin, respectively.
THE PROBLEM is that: the illumination intensity of just gel only, without any PCR product contained, still gives out data ~40, while the band intensity usually >60, GAPDH and β-actin's are ~90.
MY QUESTION is that if I should subtract the base intensity (~40) from the band intensity, then calculate the ratio.
I think many people are using this method, how do you deal with this matter???
Thanks in advance.
rick,
I am comparing mRNA expression levels of cardiomyocytes under different culture condition.
PCR products were agarose gel electrophoresed and ethidium bromide stained. The resulting band intensities were measured by means of the public NIH image processing program (Image J, http://rsb.info.nih.gov/ij/index.html). For subsequent analysis of each target mRNA, the relative expression quantity was evaluated by the ratios of band intensity to GAPDH and β-actin, respectively.
THE PROBLEM is that: the illumination intensity of just gel only, without any PCR product contained, still gives out data ~40, while the band intensity usually >60, GAPDH and β-actin's are ~90.
MY QUESTION is that if I should subtract the base intensity (~40) from the band intensity, then calculate the ratio.
I think many people are using this method, how do you deal with this matter???
Thanks in advance.
rick,
Bytheway, are here any people use ratiometric-RT-PCR to compare mRNA expression level yet?
The program I use subtracts the area between the lanes to give me the intensity, but I don't know if you have that option in your software
Dear Syaniv,
Thank you, I will check if the option is in my soft.
Bytheway, do you perform the PCR of the target and housekeeping at the same cycle number?
Oh, wait, But do you mean to subtract the area between lanes or to subtract the illumination intensity of the area between lanes?