Low RNA yield

[Tyson]

...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
|