I have dug through several threads on RG and found various opinions on this, but it appears that overall, doing qPCR comparison across tissues is messy business, because the more tissue types you have, the more difficult it is to find a stably expressed housekeeping across all tissues. Thus, I am considering simply doing a 2^-Ct expression of my data. I understand I would not have a normalization here, but this may be the best I can do (unless, of course, I ran a standard curve).
Could you all comment on this and/or provide papers where you have seen the 2^-Ct method used?
FYI, the end of the Livak et al. paper which originally described 2^ddCt states: "The endpoint of real-time PCR analysis is the threshold cycle or CT. The CT is determined from a log–linear plot of the PCR signal versus the cycle number. Thus, CT is an exponential and not a linear term. For this any statistical presentation using the raw CT values should be avoided….. Converting the individual data to a linear form using 2^-CT more accurately depicts the individual variation among replicate reactions…… Finally, statistical data should be converted to the linear form by the 2^-CT calculation and should not be presented by the raw CT values."
- I agree with Sven
- with this sort of multi factorial analysis I would screena bunch of housekeepers from your multiple tissues and look for 2-3 that cluster as stable in all tissues
- Then normalise against the geometric mean of these three housekeepers for all of your tissues/genes
- See:
Hi,
guess this would wokr, but I would rather propose to use a methodology that normalizes the expression in relation to more than one Reference gene. There are nice publications from Pfaffl et al about this. In addition you should check a bund of ref genes to find the 2-3 most stable ones to run the analysis with them
Sven Reister Thanks so much, Sven, for the answer. After doing some more digging, this does seem to be the best approach.
- I agree with Sven
- with this sort of multi factorial analysis I would screena bunch of housekeepers from your multiple tissues and look for 2-3 that cluster as stable in all tissues
- Then normalise against the geometric mean of these three housekeepers for all of your tissues/genes
- See:
- Badges
- Science method