realtime from different starting tissue masses - (Nov/22/2007 )
i would like to do some real time pcr over the life course of an insect
I would like to measure gene expression in the larvae, pupae, adult body and adult antennae.
i would probably use whole larvae, pupae and adult bodies, but the adult antennae are much much smaller amounts of tissue than the others
is it fair to do realtime on these tissues and compare gene expression across them, even though one tissue sample has a much smaller mass than the others? what are the implications of this?
of course i would be adding the same amount of extracted RNA to the cDNA synthesis, but being from different tissue masses, would i expect the cts of the housekeeping genes to vary? (some more dilute than others?) could you use houskeeping cts to normalise againsnt and would this normalisation correct for the difference in starting tissue mass?
i'm abit confused with it all so hope someone can run me through it!!
i would say, theoretically, the housekeeping gene should correct for this and also the normalizing to the same RNA amount before reverse transcription.
but how will you extract sufficient amounts of RNA from the small antennae? I mean, these are really very small and you will yield only spurious RNA amounts. i think it will be a problem to accurately quantify the RNA. Maybe with ribogreen or agilent. or are you pooling antennae of many individual insects from the same developmental stage?
and last but not least, you will have to validate an appropriate housekeeping gene, or better several ones as you are working with tissue RNA and compare gene expressions over different stages of development as well as different tissues, if I understood you correct.