Can't repeat a perfect miRNA real-time standard curve - (Jun/18/2006 )
Several weeks ago, I read a good paper about miRNA. Its experiment design is so attractive that I want to imitate their method and do some research.
According to the description in this paper, I try to get the perfect miRNA real-time standard curve showed in that paper.
I followed almost every detail of their protocol and dilute the samples.
However, after several times , I still can not get such high r2 as 0.999 described in that paper
(mine is just 0.96). And the Ct values are also a little different from them in that paper.
What should I do now?
Is there any improvement can be taken?
Any suggestion will be appreciated greatly! 
-sunnyhut-
QUOTE (sunnyhut @ Jun 18 2006, 09:01 PM)
Several weeks ago, I read a good paper about miRNA. Its experiment design is so attractive that I want to imitate their method and do some research.
According to the description in this paper, I try to get the perfect miRNA real-time standard curve showed in that paper.
I followed almost every detail of their protocol and dilute the samples.
However, after several times , I still can not get such high r2 as 0.999 described in that paper
(mine is just 0.96). And the Ct values are also a little different from them in that paper.
What should I do now?
Is there any improvement can be taken?
Any suggestion will be appreciated greatly!
According to the description in this paper, I try to get the perfect miRNA real-time standard curve showed in that paper.
I followed almost every detail of their protocol and dilute the samples.
However, after several times , I still can not get such high r2 as 0.999 described in that paper
(mine is just 0.96). And the Ct values are also a little different from them in that paper.
What should I do now?
Is there any improvement can be taken?
Any suggestion will be appreciated greatly!
R2 and efficiency always different from ideal value for real time PCR, especially the efficiency, which is more dependent on primers.
While as to R2, 0.96 is little bit lower, but still acceptable.
I use a PolyA tailed RT-PCR protocol, the R2 usually near to 0.999 using 1000pg ~ 1pg total RNA as template for high expressed miRNA.
here a link related to e and r2
http://www.protocol-online.org/forums/inde...pic=14573&hl=r2
-rye-
Rye, Thanks a lot!
-sunnyhut-