Low OD using Trizol to isolate total RNA from cell lines

Hi all,

I use Trizol to isolate total RNA from mouse cell line. Sometimes I
get a very poor 260/280 ratio (0.9-1.2). I think it might be due to
protein contamination. Has anyone there ever tried any modification to
this protocol to get a good yield and better 260/280 ratio? What
method do you use to purify the total RNA after you check with a
spectrophotometer( besides phenol:chloroform extraction from Maniatis
)?

Thanks,

Alex

[Only registered users see links. ] wrote:

We use TRI reagent (Sigma) - equivalent to Trizol, followed by an
RNEasy cleanup (QIAGEN). The yield is lower since you lose low weight
species such as tRNA, but if you're looking at mRNAs that makes no
difference. If you want to use it for RT-PCR, you'll probably still
need to do a DNAse digest, or simply design primers so they won't
amplify anything from genomic DNA.

Peter

[Only registered users see links. ] (Alex) wrote:


Don't some protocols use a proteinase K digestion, perhaps at a lower
temperature for a longer time? Just in case the purity is not where they
like it. Make sure your pH is not above neutral.

Hi Alex,

I use Tripure (Roche Diagnostics) for many years now and I have never seen
this effect. Perhaps you should use another manufacturere.

Brenda

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