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qPCR hell- any suggestions?

qPCR hell- any suggestions? - Molecular Biology Techniques

qPCR hell- any suggestions? - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 05-16-2013, 09:53 AM
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Default qPCR hell- any suggestions?



Hi
Can anyone offer any help. I am having some trouble witrh my qPCR. I run a duplex assay with 2x Taqman universal mastermix (no UNG) on an ABI7900HT and I have encountered a problem that I cant fix.

I am testing for copy number of an antibody in a qualification assay to get this test up and running for use- therefore all my samples are artifical and spiked with a known amount of target plasmid DNA, therefore i know the result I am expecting. Both the heavy and light chain antibody genes are on the same plasmid so all result should match.

When I test for the antibody heavy chain I get the expected result but when i assay for the light chain I get an unexpectedly low result. My housekeeping gene assay always works and so i don;t think the problem is with my qPCR technique.

I am beginning to suspect it is the specific target gene that is causing the issue. I have tried 6 primer/probe combinations now and always run two prelim tests to check the prime/probe sets- a standard curve against target only then a standard curve against target with my full duplex master mix.

I have sequenced the light chain gene and it is fine. The Tm of the primers are close, the amplicon lenghts are 50-150bp. The reagents are always fresh, the probes are diluted to single use aliquots to avoid freeze thaw degradation. i have used the same lot of sonicated gDNA to act as "host" DNA and thr same lot of plasmid.

Has anybody seen this sort of effect?
Can anyone recommend any other prelim testing to validate primer/probe sets before use?
Any suggestions will help?

Thanks
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