You may not have enough template sequence
Your gene of interest may not be expressed at a high level in your sample type, or the sample types you extract from yield very low nucleic acid, e.g., obtained by cell sorting, LCM, or FFPE.
For guidance on how to assess the amount of RNA input for your real-time PCR study, refer to page 25 of the Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR.
- Increase the amount of template you use in the real-time PCR reaction.
- Select an RNA isolation kit based on your sample type to maximize yield.
- Preamplify your target sequences: The TaqMan® PreAmp Master Mix Kit is intended for use with very low amounts of cDNA. Up to 100 targets can be simultaneously preamplified in one multiplex reaction. Confirm that all amplicons are amplified uniformly without bias before performing preamplification with limited amounts of samples. See the TaqMan® PreAmp Master Mix Kit Protocol for further details.
Publication: Noutsias, M. et al. (2008) Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 9(1):3