Trizol RNA Extraction Protocol: Emery Protocol w/ DNase I treatment

Tested protocols

- 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.

1. Decapitate flies on large petridish filled with dry ice, heads should then just pop off. Brain dissection: Keep flies in ethanol on dry ice, place dissected flies directly in Trizol kept on dry ice. Do not homogenize.

2. Homogenize the heads with a pellet pestle in 800 μL TRIzol (use more Trizol for substantially more sample).

3. Incubate the homogenate at room temperature for 5 min.

4. Add 160 μL of chloroform, vortex for 1-2" and incubate for 3 min at room temperature.

7. Microcentrifuge (12,000 g) for 15 min at 4°C.

8. Transfer the aqueous phase to a fresh microcentrifuge tube and precipitate with 400 μL isopropanol.

9. After 10 min at room temperature, spin down the RNA with a 15-min microcentrifugation (12,000 g) at 4°C.

10. Discard the supernatant, rinse the pellet with 1 mL 75% ethanol, and microcentrifuge again for 5 min.

11. Discard the supernatant and air-dry the RNA pellet for 2 min (Do not overdry or RNA will be impossible to dissolve).

12. Resuspend RNA in RNAse-free H2O (volume: see below DNase I treatment) and incubate at 55 DEG for 10 min to ensure that RNA is completely dissolved. Check RNA purity with Nanodrop: [260/280] 2+0.1, [260/230] > 1.

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.