Many qPCR assay suppliers only provide a general chromosomal location without disclosing sequence information. When you purchase a PrimeTime® qPCR Assays from IDT, we disclose primer and probe sequences. Here is why we think having the sequence information of your primers and probes is so important.
PrimeTime qPCR Assays are designed to avoid nonspecific amplification and have undergone a thorough BLAST search against current NCBI databases. However, with the large volume of new sequence data being generated, these databases are frequently updated with additional information. When you know your primer and probe sequences you can interrogate the databases in the future and understand your data in the context of new transcript information.
When multiplexing with several primer and probe sets in a single assay, it is critical to know your primer and probe sequences to avoid primer-primer and primer-probe interactions.
Troubleshooting unexpected data can be difficult and time-consuming, and these efforts are exacerbated when you lack sequence knowledge. Identifying why an experiment fails often requires knowledge of sequence properties—the Tm of an oligo, potential dimer or secondary structure formation, or nonspecific amplification. With sequence information, quick and accurate data interpretation is possible.
As new transcript variants are reported, knowing where your primers are located allows you to determine whether your primers will amplify the new transcript variants.
Knowing your primer and probe sequences allows you to publish according to the MIQE guidelines (see sidebar below). These guidelines call for sequence transparency so that others can better evaluate, repeat, and validate your work. Dr Stephen Bustin, one of the authors of the MIQE guidelines, notes, “Incomplete reporting of experimental detail confounds assessment of qPCR data validity, calling into question scientific conclusions that serve as a basis for further basic research and diagnostic applications” .
Knowing the sequences of your primers and probes carries important value, as demonstrated in the points above. When you are evaluating qPCR assays for purchase, be sure to ask whether you are getting assays designed against the most up-to-date sequence information, and whether you will be given the sequences for your primers and probe. With IDT qPCR assays, you will get both.