[Arabidopsis] Re: RNA extraction - Arabidopsis lyrata
(by analia_alet from intech.gov.ar)
Sat Jun 23 09:54:29 EST 2007
I've been working in RNA extraccion but with Arabidopsis thaliana.
And I used to have the same problem that you have now.
use allways fresh tissue. If you have to save it for later work, just frezze
the tissue wrapped in little packages made from Aluminium paper(role) at -
80ºC. NEVER allow the tissue to defrost. When processing it, go to the freezer
to pick up the tissue with liquid nitrogen, and be sure the eppendorfs you are
using have been treated with liquid nitrogen too. I use to pass everything by
liquid nitrogen (the eppendorffs, the Spatula, the morter...)
Other thing you can do is to use trizol instead of the quiagen kit. And then
if you want it, you can re-purify the RNA in a column from the kit.
I never used Quiagen, my favorites are Talent and Promega, and obviouslly
trizol (invitrogen or Sigma; if you want it, I have a protocol I have already
tried (and it worked))
About saving the samples in RNA later, I have never done it. Once I saved the
samples in the lysis buffer from the Promega kit, and it worked well.
Hope it serves to you, and please forgive me my english
I'm studying gene expression in Arabidopsi lyrata. I have some problems
with RNA extraction. I use RNeasy Plant Mini Kit (Qiagen) for extractions.
My leaf samples are stored in RNAlater liquid (Ambion) at -80 degrees. I
do the sample disruption with TissueLyzer (Qiagen): I put the sample,
buffer and 2 stainless steal beads in 2 ml Eppendorf tube and disrupt the
sample by shaking with TissueLyser. For young and fresh leaf samples this
works well: I get good yield of pure RNA. But older and RNAlater stored
samples are causing me problems: the RNA yield is low and it also seems
that there is some impurities in the samples (A260/A230 is really low:
between 0,2 and 1). Does anyone has an idea how should I modify the
extraction protocol to get better quality RNA? And do you have any
experience of working with RNAlater liquid? Could that cause my problems?
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