I know that if i want to see whether a certain gene of interest is under circadian control i have to collect tissue samples under constant darkness. But what i don't understand are the reasons for collecting under LD cycles. So my main question is when is it appropriate to collect tissue samples under LD and under DD? Say i already know my gene of interest is circadian and i examined it under DD conditions, then i generated KO mice, would it matter if i collect the tissues under DD or LD?
Additional question: for one of my experiments, i'm trying to determine in a transcriptome-wide manner the mRNA transcripts bound to my gene of interest, so will be doing some RNA-IP experiments at different time points in WT mice. So in this case does it really matter whether DD or LD? I would think that LD data will be more beneficial than DD as it reflects the entrained conditions in which mice in laboratories are housed under?
p.s. I'm new to the field, and the lab i'm working in doesn't study circadian clocks.
You collect samples under DD as this gives a true readout of whether your gene of interest is a circadian gene - i.e. can the oscillation free run, and is not a consequence of being driven (entrained) by the light dark cycle (as you have already alluded).
If you have already identified a circadian gene that is rhythmic under free running conditions, in any follow-up investigation of the gene you would want to keep the same free running conditions to see if your manipulation alters the ability of your tissue/gene of interest to maintain bone fide free running circadian rhythms.
If you wanted to study how photic entrainment affects your genes or is influenced by your manipulation, then you would need to study the gene in the context of an LD cycle and use phase shift paradigms etc.
Dear Haytham Mohamed,
Your question is very close to me due to the fact that I have already faсed the same difficulty. If your gene is a circadian one you have to investigate what elements of core clock engender the oscillations in gene's of interest expression, it will help you to choose CT (circadian time) appropriate for tissue sampling (when expression really exists), concerning the activity of TT-loops in LD, which is normal photoregime. Circadian time is very remarkable in gene expression profiling, the choise of it determines the result, this very choise of course depends on what you want to extract or recieve from your data.
Probably you will be interested in an alternative view on this challenge. IMHO, dynamics of expression is more informative and you may sample tissue every 4 hours, it doesn't matter wheather it is LD or DD in this case and you will have a plenty of data.
Good luck and best wishes!
Thank you all for the replies. That cleared up a lot of stuff. Good luck to you all:)
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