发信人: smileface (笑脸), 信区: Biology
标 题: Re: 请教RT-PCR的结果怎样是在线性范围内？
发信站: The unknown SPACE (Wed Nov 13 03:22:29 2002), 站内信件


no,
however,

I am not sure whether you are talking about quantitative real-time PCR
or reverse transcription PCR.


For real-time PCR, no matter how many cycles you run, you are caculating
from the cycle number that first give a significant PCR signal. So
it doesn't related with your total PCR cycles. Although you may set your
PCR reaction to 35 cycles, if your PCR give a signal at 18 cycle number,
that is linear before 18 cycles.

For reverse transcription PCR, if you care about linear range, don't run
PCR too much. You can end-label your primer and run PCR for only 20-25
cycles. This is as sensitive as real-time PCR. And you should also
have a positive control with serial dilution of plasmid DNA.




【 在 agarose (agarose) 的大作中提到: 】
: 请教RT-PCR的结果怎样是在线性范围内？
: 是看随cycle 数目增加，beta-actin的量也增加，在此线形范围内，目标基因表达有差异
: ，才有意义，对么？
: 那么说是每个试验都要找线性范围么？
: 怎么办呢？守在cycler旁边，过一个cycle抽去5ul的样品去跑胶么？
: 多谢指点


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