I am planning to extract RNA from Drosophila melanogaster heads, which will be used for RNA sequencing to determine transcriptional changes across specific mutants. Methodologies for RNA extraction vary widely between labs. With the intention of RNA sequencing to find transcriptional up- and downregulations, what experience do you have in terms of methods/protocols/kits for RNA extraction from Drosophila melanogaster heads that have worked well for this purpose?
If you have enough tissue, Trizol (or similar) works great. If you are limited in terms of starting tissue, kits usually work better. I have worked with Promega and Qiagen column kits, you will need to test them to find out which one works better for your tissue.
Good luck!
trizol and then pass to RNeasy mini or micro kit from Qiagen . It comes very clean. I used it for RNAseq on ring glands. Cheers
If you have enough tissue, Trizol alone is the best option (in my opinion) because it is the cheaper by far.
Regarding purity, Trizol will give you very clean (no other chemicals like phenol or guanidinium salts) RNA since you can do as many chloroform and 70% ethanol washes as you wish. Again the real limitation here is starting tissue amount (that correlates with total RNA content) since you loose a little bit of RNA with each wash.
If you have limited amount of tissue (that means not too much total RNA), column kits are the best option using the kit buffers or combining with Trizol as Dr. Di Cara suggests.
In terms of RNA quality (no RNA degradation during the process) both methods work fine. You can check this by measuring RIN (RNA integrity number) with agilent bioanalyzer or tape station (first one is better). It ranks from 0 (total degration) to 10 (perfect RNA, no degradation). However, RIN = 3 is good enough for PCR and RIN > 7 (better > 8) are usually recommended for RNA seq. Again, total RNA quantity matters so keep in mind that if you do not have enough RNA RINs will be bad even if there is no RNA degradation.
Good luck and let us know if your experiment works!
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