Low RNA yield

pathos pathos at freerealtime.com
Fri Feb 4 16:36:22 EST 2000



Yes possibly and since she is on the high end of the amount of cells
recommended by the manufactuer of the kit, she is in danger of not having
enough RNase inhibitor.  She should use more of the homogenation buffer
which should have lots of Guanidinium isocynate in it.

In article <WhIm4.4684$45.227122 at news2.rdc1.on.home.com>, "Tyson"
<tyson at canada.com> wrote:

> ...perhaps there is RNase contamination somewhere eg. pipette tips etc..
> 
> Tyson
> 
> 
> "Karen Roberts" <leehrat at mindspring.com> wrote in message
> news:3898A662.12D138BA at mindspring.com...
> | Hi,
> |
> | I am posting for a technician friend who doesn't have Internet access
> | (one of the few).
> |
> | She says that she is getting low yields of RNA from thymus cell
> | culture.  She uses Qiagen RNAeasy kit.  The cell number in her samples
> | is 1 x 10(7), which is the max starting amount recommended.
> |
> | She has repeated the assay three times and it appears to be a systematic
> | problem.  She says she is careful to homogenize the cells well acc. to
> | protocol; in short her methods are fine.  Though she didn't run an RNA
> | gel to see how abundant it is, she did do RT PCR for B Actin and got a
> | signal, but it was much less intense than what is usual for Actin.  So
> | she does have RNA just a low amount.
> |
> | She then made fresh buffer from the kit with fresh B 2ME and still got
> | the same results.
> |
> | Could it be the kit has gone bad?  She received it new from Qiagen in
> | September.  IF not, what else could she try to increase her yields.
> |
> | PS. This assay was done by another tech shortly after the kit arrived in
> | Sept. and she had the expected amt of RNA.  Though that would suggest
> | it's my friend's technique, she asked the investigator to repeat it and
> | she also got low yields.
> |
> | Ideas appreciated.
> |
> | TIA,
> | Karen
> |

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology




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