Trizol RNA extraction of bodies, Flychip Protocol

Published on Author Anne Petzold

Protocol

“Small scale extraction of total RNA from Drosophila melanogaster” @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf

(extraction based on combination of Trizol and Chloroform)

Samples

app. 30 male fly bodies.

Process

After incubation @ -20 DEG (step 12), sample was split in two. Sample 1 was temporarily stored @ -2 DEG in 500 ul ethanol, while sample 2 was treated with DNase I.

DNase I treatment (following manufacturer’s instructions):

– resuspend 10 ug RNA in 1x DNase I reaction buffer to final volume of 100ul

– add 0.5 ul of DNase I, mix, incubate @ 37 DEG for 10″

– add 1ul 0.5 M EDTA (microfiltered and autoclaved EDTA)

– heat inactivate @ 75 DEG for 10 “

– resume original protocol, step 12 (spin down RNA)

Results

Sample 1 (no DNase I treatment): [260/280] 1.75, [260/230] 0.73, 489.7 ug/ul (Nanodrop, RNA-40 mode)

Indicative of contamination with phenol or protein – can I still use it? How to best store it? Now stored @ -20 DEG. Probable sources of contamination: #1 pestle, other: vortex longer when chloroform extracting to get rid of proteins, homogenize more, here some tips: http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-do-s-and-don-ts-of-total-rna-isolation.html.

Sample 2 – did not produce a pellet after step 12, discarded