relative expression of genes using semiquantitative PCR - Semi-quantitative PCR (Jul/05/2011 )
First of all would like to apolgise for posting a question on semi-quantitative PCR in the era of real-time PCR.
I have analyzed expression of three genes: a, b and c in certain pathological tissues. While a is a house keeping gene, b is the gene that has got upregulated in the pathological condition and c is the gene that has got downregulated. I have performed agarose gel electrophoresis and have the gels analyzed by Bio-Rad gel documentation system. I have the intensity/density values for all the genes studied from 10 subjects. How do I analyze my results? i have seen some papers which say that they have seen relative expression with respect to the housekeeping gene. Few others say, they have taken the values of the house keeping gene as 100%.
Can anybody suggest how to compute the expression levels of genes b and c with respect to a.
Thanks in advance,
If the intensities are linear (not logarithmic), which I think they are, I would just do b/a and c/a on the intensities for calibrator (control sample) and the same for different pathological samples. Then sample1/calibrator, sample2/calibrator and so on for both genes to get the normalised relative semi-quantitative ratio with calibrator as 1 (= 100%).