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qPCR with low efficiency,HELP please!:)


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#1 anfernee

anfernee

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Posted 17 November 2009 - 04:22 AM

Dear folks

I have been running qPCR to test the expression level of 4 genes of interests.Now I am stuck because the efficiency of PCR is low (60~85%).Sometimes I got back very nice efficiency more than 90% which I considered can be used for analysis but it is not reproducible between batches of runs...The thing is these four genes are in one gene family so there is no much room for me to redesign optimal primers. (like GC content between 40~60%,Tm 55~60 oC etc) I have read some threads saying when using cDNA to build the standard curve it works well but when using plasmid the efficiency declines.Is it due to the inhibitor in the plasimd? But I have been using very much diluted plasmid DNA (starting from 50pg and 10-fold dilution *7) so I would not consider this point...I am using Tm of 57.4 oC based on gradient PCR.

I am using Biorad IQ5 and the mastermix they provide for SYBR.Because the primer dimer issue (in one gene of four) I add one step into the program of collecting fluorescent signal at 75 oC (15s) rather than at 72 oC (30s) for extension.Will the competition between target amplicon and primer dimer affact the efficiency?

To summarize:
1.PCR efficiency is low.
2.Did I do it right for the primer dimer issue?
3.What can I do for both issues considering I do not have much choice for my primers?

Thanks in advance for any reply to help!!!




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