Disagreement between triplicate results in Q -PCR - (Sep/08/2011 )

Hi everyone,

I am new to Q-PCR.
Recently, i have been using SYBgreen Q-PCR to check the relative gene expression . For every samples i did it in triplicate, however, i always get a disaggrement or large variations between the results of my triplicate trials including a difference of Ct value and a non-smooth curve in the amplication plot within a sample with triplicate trials.

I was just wondering how i can improve the smoothness of the amplication plot of those triplicate trials of a sample and what tricks you guys are using in order to have a nice Q-PCR result. I am sure that i have pipetted an equal volume of mastermix into each sample in the eppendorf tube and then into the 96 well plate. And i am sure that i have mixed well the solution by pipetting up and down for about 7 times. Yet, i still get a bad result....

Pls kindly give me some suggestions or tips..

THX everyone!!

What volume of sample are you adding to each reaction? And have you calibrated or at least checked the accuracy of your pipettes lately?

I added 10 ul for each reaction. does this have the relationship?

Well i have not checked the accuracy, um.. i dun know but i think it's ok

The reason I asked about the sample volume is that sometimes if the volume is too low, and your pipettes aren't brilliant, the variation in amount of template between replicates can be quite significant.

What kinds of differences in Ct are you getting between triplicates?

The Ct Difference I get is about 1 to 2 cycles. Is it too large?

MurphysunHKU on Sun Sep 11 23:45:30 2011 said:

Difference of 1 to 2 cycles in what exact Ct value? It's normal to get such high variation in later cycles (like 33) but certainly not around say 20.

You say you added 10 ul of sample to the reaction of what final volume? If it is a cDNA in RT mixture, recommended maximum sample volume is 10% of the final volume. So unless you have a 100ul reaction, that's way too much.