1 reply to this topic

[mercury]

Hello all,

I have some basic queries about qPCR data analysis-
First of all let me explain you the experimental set up. I use human embryonic stem cells and differentiate it to various tissue specific cell types. Along the process the gene expression changes drastically. To study this, I will utilize qPCR on our ABI-7500fast system. For basic understanding my questions are-
a) What should be the calibrator for the study? For instance, when I have undifferentiated (ES) cells and differentiated cell from different days (e.g. Day0, day5 day10 day15 and day20), in this scenario there are certain genes highly expressed in undifferentiated (day0) cells and some vice versa. If I calibrate it with Day0, I lose the one important sample from the experiment in the resultant graph.(which is very important foe the genes highly expressed in that point)
I saw some publications in which they have used relative expression, by which they converted the maximally expressing gene to be considered as 100% and relative to it the other ones. Is it the right way? Because some time it give weird results, for instance in the above set up genes absolutely being absent can show out some peak.
c) What will be the ideal condition of make the data in a graph (plotting RQ, ddCt etc?)

Please help!
Regards
m

[Trof]

a) I would think that day0 is the natural calibrator. Only requirement is that the there is always some expression for all the genes you want to calibrate, if the gene is absent you can't calibrate it.  You need to decide what you actually need to compare and choose the calibrator accordingly. If you compare differentiated to non-differentiated, then the day0 is a clear choice.

c) You should plot normalised ratio, if the differences in expression are big, several folds, maybe you should try using logaritmic scale.