You may not have performed bioinformatic validation of your sequences

This section applies to TaqMan® assay design and SYBR® Green assay design.

Custom TaqMan® Gene Expression Assay design is based on a template sequence provided by the customer. You can also design your own assays using other programs, and send the oligonucleotide sequences to Applied Biosystems for synthesis. You must perform bioinformatic evaluation of the sequences prior to submission to avoid PCR failure.

Read Bioinformatic Evaluation of a Sequence for Custom TaqMan® Gene Expression Assays to understand how to run a bioinformatics evaluation of a template sequence.

Bioinformatic evaluation allows you to accomplish three tasks:

  1. Identify a unique sequence by using BLAST (basic local alignment search tool). If the sequence submitted is unique (or from a non-homologous region), the assay that is designed on this template only detects the transcript of interest.
  2. Mask low complexity regions by using RepeatMasker. Low complexity regions, such as ALU sequences, are usually found within genomic DNA sequences. If you design your assay over these regions, the primers or probe will be depleted quickly if there is any genomic DNA in the samples.
  3. Mask SNP sites in the sequence, so that primers and probes will not be designed over SNP sites.

If you performed bioinformatic validation correctly, you may have problems with reverse transcription.

Reverse transcription (RT) is the process by which RNA is used as a template to synthesize cDNA. The single stranded cDNA molecule serves as a template for the real-time PCR.  Different RT methods can have significant impact on real-time results. The RT reaction is often overlooked when troubleshooting late CT values (>35) or when no amplification is observed. Considerations have to be made for the type of RT reaction that was performed (one-step or two-step), the linearity of the RT reaction, and the range of RNA input (capacity) the RT chemistry will tolerate.