# How to design relative quantification study? - (Nov/27/2007 )

Hi all,

I am somewhat new to real time PCR and am having a little difficulty trying to get my head around the concept of relative quantification as it applies to my particular study. I have tissue samples from mice of different genotypes (including wildtype) at 4 different time points (including a zero point). I want to compare both within genotype changes with time, and between genotype differences at each time. For the within group comparisons, obviously expression levels will be relative to the zero time point. My problem lies with the between group comparisons. Do i simply compare levels for each time point relative to the wildtype zero time point? Or would it be better to calculate levels for each time point relative to the respective zero time point for that genotype and then make a between genotype comparison of mean % increase at each time??

I'd appreciate any advice/comment.

Hi,

I'm doing very similar experiment.

First you were right about comparing timepoints against zero sample. (computed from relative ratios housekeeping-gene of interest)

Second, I would compare the relative ratios of each timepoint of different genotypes to the corresponding timepoint of wild-type, i. e. in time zero, compare all genotypes to timepoint zero of wt, timepoint 1 compare to timepoint 1 of wt etc.

I'm doing very similar experiment.

First you were right about comparing timepoints against zero sample. (computed from relative ratios housekeeping-gene of interest)

Second, I would compare the relative ratios of each timepoint of different genotypes to the corresponding timepoint of wild-type, i. e. in time zero, compare all genotypes to timepoint zero of wt, timepoint 1 compare to timepoint 1 of wt etc.

That makes perfect sense! Many thanks!