• You may check the template used in primer design was DNA or mRNA sequence. If it was a DNA template, the primer(s) sequence may be from intro, which will make the PCR of cDNA not work, since cDNA is from reverse transcription of RNA template. For PCR of cDNA, the template for primer design should be mRNA. If you are going to detect gene expression, the primers should be across the exons.

What was the template that was provided to Primer Premier 6 Van Phung ? Was it the genomic DNA sequence of the gene, or just the spliced mRNA sequence? If it was the former, then that answers why you saw amplification with the plasmid and DNA, but not with your cDNA.

Did you control the extraction of rna and rtpcr? You did not talk about RT-PCR control, however this problem can involve from many thing, it is better to do all the steps again more carefully, RNA extraction, cDNA synthesis and RT-PCR, if the problem is not solved you need a new primer that it has been designed for the target mRNA sequence in the exon region.

RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. Van Phung

I'm guessing your primers do not match to the cDNA for your gene. Your primers have to be in exons or UTRs for qPCR. Other regions (promotors, introns, intergenic) are either not transcribed or get spliced out.

Try designing them again.

Good luck!