QPCR - (Aug/12/2008 )

Dear colleagues,

I have performed QPCR to measure the expression levels for specific mRNA, and when i got the results there were no differences in the Ct values for the transfected cells and the the untransfected cells, that means there was no expression for this mRNA, and i proved that by running some of the QPCR reaction on agarose gel, the amplicon size that i was expecting is 700 bp. However, when i performed a normal PCR using the same cDNA that i used for qPCR with the same primers for 25 cycle i got a very strong band with the expected size.

So please if anybody have an i dea or explanation for this.

Best regards

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Try changing your annealing temperature. Real time machines seem to need different Ta than bog standard thermocyclers.

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You mean try lower annealing temp?????

Thanks

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Sorry, but I dont quite follow you.

Did you get a signal with your qPCR? How high were those Cts you mention?

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I got ct values around (29) in the transfected, untransfected and nontemplate control, qPCR was performed for 40 cycles. After that, i ran those reaction on a gel and i could not detect any band, however, icould detect the right bind when i performed normal PCR for 25 cycles.

Thanks
any signl

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Yep. Although if you are getting a bunch of garbage non specific amplification then you might need to increase your Ta. Don't forget to wear your lucky socks too.

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And try to design oligos that will give an amplicon of about 100-200 bp. 700 is too big for qPCR.

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Madrius is absolutely right:
700bp is much too long for qpcr.
Maximum for most master mixes is around 200bp, only few can do it with longer amplicons... and then you should prolong the amplication time

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Hi,

I designed two PCR primer sets one with primer express (22bp-final product = 63bp) , one with primer 3 (20bp final product = 80). and also used a previousely published set (21&25bp- final product 134bp). I got best band with 5.0 amd 5.5 mM MgCl. is it too high for PCR ? what is the maximum range? I heard it is only up to 4mM one can go up with MgCl

the problem is, with lower concentrations of cDNA (0.1, 0.01) the curves amplify inconsistently in all 3 primer sets. curves at these low concentration come up at different CT values. I think this due to the low mRNA concentrations at lower concentrations. I tried doubling the primer concentration then efficiency is so high, and couldnt get a good standard curve. Can I use 0.5 or 0.4 dilutions??

Are there any other ideas? can I increase annealing Temperature ? now i am using 60C?

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Try adding some carrier DNA (like 10ng/uL of plasmid DNA that doesn't contain your gene) - that is dilute your cDNA in a solution containing other DNA.

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