Technique / Molecular Biology / RNA transcriptional and post-transcriptional regulation


RNA extraction



RNA extraction

I have isolated total RNA from non adherent cells with the TRIZOL reagent.
At the end of the procedure, I solubilized the pellet with DEPC-treated
water. After this, I measured OD 260 and 280 and calculated the ratio
260/280. This ratio should be 1.6 to 2.0. Sometimes I have got around 1.6
but generally 1.4 to 1.5 ... it is too low but the RNA is OK on agarose gel ... ...

Last update 06-Oct-2002, Rating of 1 votes.

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By kar on 15-May-2008
Your high A260/280 is probably due to protein contamination. I generally go for 1.8 - 2 and a A260/230 (salts) of 2 or above.

Are you using a kit, or chloroform extracting?

For chloroform extraction: Generally, I can achieve this hassle free by pipetting the aqueous phase very carefully after chloroform extraction - without going too close to the DNA interphase.

Further RNA purification (as I need very high purity for Microarrays) can also be achieved by LiCl precipitation, which is specific for RNA and not protein (hence lowering your A280 reading).
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By prafulla on 06-Oct-2002
I have isolated RNA from bonemarrow cells of swiss mice using Trizol reagent and had solubilzed the RNA pellet in millique water.I have got the degraded RNA.can i have the trouble shooting for my preparation
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