发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 检测基因表达变化时，RT-PCR和Northern blot首选哪个？
发信站: The unknown SPACE (Sat Dec 7 22:42:30 2002), 站内信件

if you are look at splicing events and you definitely would
like to use northern blot, though technically it might be
difficult. RTPCR is more sensitive for quantitation of total
messages, or you can actually design primers for alternative
splicing products to specifically measure them in a total
population of mRNAs. i like RTPCR since it is quick to roughly
estimate changes for steady level of RNA. and sometimes i do
semiquantitative RTPCR, which means that i stop the RXN before
it reaches the plateau, do a following southern, or even
incorporate radioisotope during the PCR process. Realtime is
fine, but you have to normalize that to certain internal control
messages, e.g., actin, tubulin. Realtime uses fluorescence dye
and tells you when the signal could be detected. for example,
if tubulin for both samples are detected @ cycle 18, and then
for the gene you are interested, one of them @ cycle 20, the
other @ cycle 24, then there is 4 cycles of diff. but it is
not linear diff. the internal control should be carefully
picked up, think about the controls for microarray. experimental
conditions might change the metabolism of cells and thus affects
some "housekeeping gene".

【 在 clonist (chill out) 的大作中提到: 】
: in terms of miRNA people use northern. i didn't claim it's
: always right. just try to say nothing is absolute in biology
: 【 在 smileface (笑脸) 的大作中提到: 】
: : clonist //hehe, take it easy.
: : generally, RT-pcr is the most sensitive assay to RNA, although sometimes
: : it cause pseudo positive.
: : However, RT-PCR can't detect isoforms, or alternative splicing products.
: : And because of its pseudo positive.
: : a northern is necessary.
: : However, S1 mapping of nuclease protection assay is roughly 20 fold more
: : sensitive than northern. Although these two methods also can't give
: : you the information of the full length or some isoforms' information.
: : all in all, these assays are complimentary to each other.
: : Depends on your aim, your project, what you have at hands.


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