The input amount of RNA is not optimal for the size of RT reaction

The assay design could cause later than expected amplification if the assay being used does not detect all of the splice variants for the gene of interest.

Solution:

Source of AssaysYou need to:
TaqMan® Gene Expression Assay

Check the alignment of the assay with target sequence. When you search TaqMan® assay, there is an Alignment Map link under each assay ID when search result is returned. The link leads to an alignment map as shown below:

  • The online help explains how to view the Alignment Map.
  • Choose an assay that will detect all of the splicing forms of the gene.
Custom TaqMan® Gene Expression AssaySubmit a template sequence that is common to all of the alternative splicing variants.
Assay designed by yourselfDesign the primer and probe on a template sequence that is common for all of the alternative splicing variants.