RNA extraction

Robert Hartley rh at mblab.gla.ac.uk
Fri Jul 21 09:50:23 EST 2000


In article <vkPb5.1011$WM6.75101008 at news1.mtl.metronet.ca>, "Tina"
<anfortin at webnet.qc.ca> wrote:

Hello Tina.

> Hi everybody!
> 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
> (no apparent degradation). 

Istly I normally check the RNA ratios in H2O. Secondly I had been using
RNazol TRI etc but now I use simple home made reagents.

4M GITC
50mM EDTA
0.5% n-laurylsarkosine
sometimes BME

lyse using this and you can even freeze your samples down for later.

Then I use acid phenol chloroform IAA and a standard Isopropanol PPT.

2X EtOH wash

Resuspend in H2o and do the spec in H2O
As for the SPEC. I use a 224-320 wavelength scan 260/280 and 230.


This should give you allthe data you require. I beleive that TE will lower
the ratio. I dont know how much and I can't remember the ref.

>Which step of the Trizol method can I improve to
> increase this ratio? I have already read that to calulate the ratio it is
> better to take ODs of RNA in TE buffer but other people have already got
> good ratios with RNA in water.

When I did use things like Ultraspec i did an Acid Phenol chloroform
second extraction with the aqueus phase.

One thing that als oaffect the spec is the pH of the water you are using
for resuspending.

This is where I go out on a limb. :-)

dH20 has a lower pH than 7 i think it is about 6.5 DEPC h2o is lower than
that at about 6.

Thats why you see refs to alkaline H2O and why some people use TE at pH8ish

Try using H2O at a pH of at least 7.

cheers
Bob; Very Sunny Scotland






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