- Phenol-Chloroform extraction from F Schweisguth @ http://francois.schweisguth.free.fr/protocols/Extraction_Drosophila_total_RNA.pdf
- Chloroform extraction from FlyChip @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf
- P Emery. RNA From Drosophila Heads, From: Methods in Molecular Biology, vol. 362: Circadian Rhythms: Methods and Protocols. E. Rosato [ed] © Humana Press Inc., Totowa, NJ
Settled for the Emery Protocol for simplicity, yield and success.
Emery Protocol for RNA Extraction from Drosophila Heads (for ~50 heads or ~8 bodies)
Perform in the cold room up to DNase treatment.
DNase I treatment (@labs.genetics.ucla.edu/lusis/dnasetreatment.htm)
Theoretically, below values are for 1 ng of RNA/ul, but I use the same volume of DNase I to good avail, depending on type of sample and do not fine scale for concentration as by Nanodrop.
|Brains||Heads||Bodies (n=5)||Bodies (n=10)*|
|RNA in x H2O||10 ul||30 ul||50 ul||100 ul|
|10x DNase I RB||1 ul||3 ul||5 ul||10 ul|
|DNase I||1 ul||3 ul||5 ul||10 ul|
|25 mM EDTA||1 ul||3 ul||5 ul||10 ul|
*If more sample collected than needed for PCR, just dissolve the RNA pellet in the appropriate amount of RNase-free H2O, take an aliquot of the dissolved RNA and just perform DNase I treatment for the small volume. Keep in mind that cDNA produced from this RNA will be diluted further and only about 1 ul of this diluted cDNA is enough for 1 PCR reaction.
- Prepare a DNase I treatment mix (Without EDTA!).
- Add to each RNA sample and incubate RNA @ 37 DEG for 15"
- Add 25 mM EDTA to each RNA sample and incubate @ 65 DEG for 15"
- Collect reaction by brief centrifugation and immediately place on ice. Can be used straight away for amplification or reverse transcription.