Low RNA yield

Karen Roberts leehrat at mindspring.com
Wed Feb 2 16:49:22 EST 2000


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.


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