Redundancy of real time PCR results - (Oct/14/2008 )
hey guys, I might need you guys opinion regarding on real time PCR results.
I did some real time PCR run (1 step run). I used 2 different dilution: 10 ng and 100 ng of RNA. Interestingly, I found the gene was upregulated from 100 ng BUT not from 10 ng. Why so? I mean...... how is it possible? Does dilution make that big difference? Thanks in advance!
-timjim-
QUOTE (timjim @ Oct 14 2008, 04:55 PM)
hey guys, I might need you guys opinion regarding on real time PCR results.
I did some real time PCR run (1 step run). I used 2 different dilution: 10 ng and 100 ng of RNA. Interestingly, I found the gene was upregulated from 100 ng BUT not from 10 ng. Why so? I mean...... how is it possible? Does dilution make that big difference? Thanks in advance!
I did some real time PCR run (1 step run). I used 2 different dilution: 10 ng and 100 ng of RNA. Interestingly, I found the gene was upregulated from 100 ng BUT not from 10 ng. Why so? I mean...... how is it possible? Does dilution make that big difference? Thanks in advance!
Hi
A 10-fold dilution is quite a lot, if you have a rare target. I'm not so familiar with one-step reactions, as I normally do the reverse transcription prior to Real time PCR (two-step). On a standard curve with 4-fold dilutions series of cDNA, you can really se that dilution of your template matters.
Maybe you can make a "standard curve" for you type of samples - as I'm guessing the reason for using 10 ng is because your sample material is difficult to come by.
Kirsten
-Kirstenf-
QUOTE (timjim @ Oct 14 2008, 05:55 PM)
I did some real time PCR run (1 step run). I used 2 different dilution: 10 ng and 100 ng of RNA. Interestingly, I found the gene was upregulated from 100 ng BUT not from 10 ng. Why so? I mean...... how is it possible? Does dilution make that big difference? Thanks in advance!
What were your Ct values for the samples? Some people take Ct as valid only if it's between 20 and 30, co you may trust more the value from this range.
-Trof-