I want to do RT PCR of mRNAs of Heat Shock Proteins related to Drosophila tissue system. What is the procedure of sample preparation for RT PCR in insect tissue system?
I suggest to use Sigma Aldrich TRI reagent for total mRNA isolation. We tried many different silica membrane and silica pearl based RNA isolation kits, but there were not working so efficiently as TRI reagent, which is a phenol containing solution. 10 animals (10 larvae, 10 pupae or 10 adults) are sufficiently for a very high yield (but I could isolate from 15 larval brains as well). Animals frozen by liquid nitrogen can give better yield. After the RNA isolation you should transcribe your mRNA into DNA (copy-DNA, cDNA) using reverse transcriptase, random hexamer primers and dNTP (ex. Thermo Scientific Revert Aid RT kit). After the reverse transcription you should eliminate the remained RNA by RNase treatment. This way practically you will have a cDNA sample representing all mRNA transcribed in a given tissue or animal. With your gene/transcript specific primers you can check the amount of certain transcripts by RT PCR.
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