I have some problem with gene expression analysis with qPCR.
It is my first time that I am using qPCR, as a result I have some problems which may be so basic and I would be so grateful if you could help me to deal with them.
I used Gapdh as endogenous gene. I used cloned gene for making the standard curve. I used from 1000fg to 1.6fg and use dilution rate of 1:5.
Unfortunately, the result of qPCR showed improper amplification curve with very low standard curve efficiency. I changed the baseline from 3-15 to 3-8 and I got better curve but again very low standard efficiency.
Dear Kathryn,
I started with higher amount of template previously. From ng and i did not get good results.
In your idea is the problem amount of template?
I would say the opposite. I think you are still getting very rapid amplification. The first plot where you have some wells that curve back down is commonly seen with highly abundant genes like 18s when amplification is happening during the baseline phase. What does the Rn plot look like? I would also look at the instrument raw data pane to see what is happening in each well.
Thank you, for your suggestions.
I should mentioned that previously I used higher concentration, startrd from 5 ng.
And in different experiment i decreased the concentration, because i got the amplification curve so early from 5th cycle. As a result it seemed that higher template concentration is not the source of problem, in my idea.
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