Why do I see low, poor, or sub-standard amplification efficiency in my qRT-PCR assay?

The template that you chose to use in generating your standard curve may not express your gene of interest abundantly enough to be detected after the several 10-fold serial dilutions required for the standard curve. In such a case, many of the standard curve reactions should be yielding high Ct values (> 30). You can lower the serial dilution factor to 2-fold, and generate a new standard curve. You can also try using an alternate source of template for the standard curve reactions, such as cDNA derived from a universal source of RNA, cDNA derived from a full-length in vitro transcript of the gene of interest, or even a full-length cDNA clone of the gene of interest.

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