TRIzol responses

Karyn Lynn klynn at STUDENTS.WISC.EDU
Thu Jan 25 15:27:15 EST 1996


Since so many people asked me to share the information on TRIzol, following
are the responses I received:

--the TRIzol reagent worked very nice for the isolation of RNA from
Arabidopsis. However, I never isolated mRNA, I performed the reverse
trtanscription directly with total RNA and it worked very nicely. Also the
subsequent PCR using oligomer primers worked well. I have never compared
this method with the 'regular' phenol/chloroform protocol for these
experiments, so I cannot say anything about the differences. The protocol
works nicely with small amounts of tissue (<1g fresh weight) and it is much
quicker than other protocols. However, a student in my lab has compared the
TRIzol method fo total RNA isolation with the phenol/chloroform method with
Arabidopsis and found no significant differences when he performed Northern
blots afterwards. Also the recovery was about the same for both methods. So
my recommendation would be to use TRIzol only if you have quite a number of
samples and if you have small amounts of fresh weight, otherwise you might
also use the traditional method.

--The TRIzol worked really well for isolating RNA from Arabidopsis.
It is very easy to use and can be scaled down to do the whole procedure in
Eppendorf tubes.

--I have used Trizol reagent and got very good success with it. I was using
the reagent on tobacco leaf tissue. The total RNAs were used in Northern
and RT-PCR. I got good results from both Northern and RT-PCR. The gene I was
studied is a subunit of Mg-chelatase (chlorophyll biosynthesis pathway) so its
mRNA are present at reasonable amount within green leaf. I did not compare
the Trizol reagent to the normal protocol.

--I have had mixed results with TRIzol reagent in RNA extractions.  It
largely depended upon the tissue sample.  In young green tomato fruit it
worked resonably well.  However, in later stages of development (ripening
fruit) the RNA preps were useless (degraded).  In both cases there were
problems with copurification of some polysaccharide material.  However,
when I went to the more traditional purification procedure
(phenol-chloroform) I had excellent results.  I can only offer that you
should try a small scale TRIzol prep and see what you get.

--I think that the TRIzol Reagent is more suitable for animal cell
culture or blood, since there is no grinding step of the material. Someone
in my lab tested this reagent, he obtined a poor yield RNA and he could not
perform PCR on it...
        Personnally I've used a smal scale "hot" phenol/chloroform method
(see Verwoerd et al., 1989 N.A.R. vol.17 : 2362)  to isolate RNA from down
to 200 mg of Arabidopsis material (with a yield of 80-200 microg) and
performed successfully PCR on the 1st strand cDNA.

--We have used Trizol to isolate RNA from  Arabidopsis inflorescences, it
worked fine although the RNA tended to be a bit dirtier than the usual
Guanidinium thiocyanate protocol, to get round this we did extra
phenol/chlorform extractions.  It is much quicker than the guanidinium
thiocyanate protocol and can easily be carried out on small samples.We used
the RNA for Northerns.
We have also used the Quiagen RNA isolation kit which worked well too.

And from Life Technologies:

--Regarding the use of Trizol with plants,

  We have purified RNA from poinsietta leaf as follows:  pinch off tip and
allow
  exudate to form.  Homogenize 15 mg (total weight) leaf with ground glass
  homogenizer in 1 ml of Trizol.  (In general, 1 ml of Trizol is used for
50-100 mg
  of tissue.)  Follow with regular protocol.

  Actual yields from plant leaf :
   Poinsettia: 33 ug RNA from 45 mg tissue (0.7 ug/ml)
   Tobacco: 76 ug RNA from 100 mg tissue (0.8 ug/ml)

 -If a sample is known to have a high content of proteoglycans
  and/or polysaccharides (such as rat liver, rat aorta, plants), the
following
  modification of the RNA precipitation step should remove these
  contaminating compounds from the isolated RNA:
   Add to the aqueous phase 0.25 ml of isopropanol followed by 0.25 ml of a

   high salt precipitation solution (1.2 M sodium citrate and 0.8 M NaCl)
   per 1 ml of TRIzol used for homogenization.  Mix the resulting solution,

   centrifuge, and proceed with isolation as described in the protocol.
   The modified precipitation effectively precipitates RNA and maintains
   proteoglycans and polysaccharides in a soluble form.  This procedure
   should ONLY be used if the sample is known to have a high content of
   proteoglycans and polysaccharides.  To isolate pure RNA from plant
   material containing a very high level of polysaccharides, the modified
   precipitation should be combined with an additional centrifugation
   of the initial homogenate.

 -Starting with 1.8 g of Arabidopsis tissue (whole plant),  2 mg of total RNA
was purified with Trizol Reagent.
 Using 2 selections with the Messagemaker mRNA Isolation System, 11.5 ug of
mRNA was recovered.

Sincerely,

Sharon McRae
Life Technologies
Technical Services
smcrae at lifetech.com





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