发信人: favor (MIG25), 信区: Biology
标 题: Re: Real-time pcr:standard curve
发信站: Unknown Space - 未名空间 (Tue Jun 8 16:10:36 2004) WWW-POST

1) Do your realtime primer/probe span exons? If not, genome contamination may
cause the discrepancy.

2) Narrow down your standard curve range, for example using a two or three
times dilution intead of ten.

3) It seems you use absolute standard curve method. If you just wanna compare
two samples, why not use total cDNA make standard curve and normalize to
housekeeping gene?



【 在 harrywfu (天南海北山东人) 的大作中提到: 】
: Using northern blot I got about 3~fold difference in mRNA expression between
: two samples. When I switch to real-time PCR, I only got 80% difference.
: Basically I transfect luciferase expression vector and check luciferase mRNA
: level. To make standard curve, I use dilution 1-10-100-1000-10000 of
: pGL3-promoter plasmid.
: Is there any suggestions?
: I am thinking:
: 1.Is that OK to use DNA to make a standard curve???
: 2. THe efficiency of reverse transcription. Probably due to low efficiency??

:
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