发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: RNA extraction
发信站: The unknown SPACE (Thu Oct 3 21:53:00 2002), 站内信件

the essential step is to keep everything as clean as possible.
and do not use anything you are suspicious. Ambion is a good
company that has very good experience dealing with RNA. my
personal experience is that: ALWAYS aliquot reagents. i used
to use DEPC'd ddH2O, but now i would rather use commercial
nuclease free water. try to be a quick and clean hand, quick
and dead, you know that? i.e., you quick and RNase dead, no
no vice versa. when you deal with different biological samples,
you have different approaches for the best isolation and
recovery of RNA. however, the most important thing is to
treat the sample ASAP. do not assume that you can isolate
good quality of RNA from frozen tissues. typically, ppl use
liquid nitrogen and throw the tissues into TRI reagent that
denatures protein including nuclease. Ambion has an RNA
Stabilization reagent, i have not used it, and it seems that
it has high content of salt and it is pretty effective. besides
TRI, Qiagen has several kit for isolation though most of them
are pretty expensive, Clontech has a good one for isolation
of RNA for microarray. sometimes the RNA isolated using TRI
is not good for microarray, maybe due to moiety of phenol.
for nuclease enriched tissues, you MUST treat the samples w/
enough rounds of phenol/chloroform after primary TRI isolation.
well, the final step is to dissovle the RNA. do not let it
dry too much. and remember that you have to treat the RNA
with RNase free DNase, during which the RNA could be degraded
essentially in the presence of RNase.



【 在 loveyu (小呆鱼) 的大作中提到: 】
: 最关键的一步是什么，怎样才能成功地提出来呀。
: Thanks


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