FAQs on Real-Time RT-PCR

PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). There are several ways to search for primers: by GenBank Accession, NCBI protein accession, LocusLink ID, PrimerBank ID or Keyword (gene description). PrimerBank contains about 180,000 primers covering most known human and mouse genes.

Polymerase Chain Reaction Amplification (PCR) is one of the most actively used techniques in molecular biology. In recent years, PCR has been increasingly used for gene expression detection or quantification. It is a more convenient method in gene expression studies comparing to other techniques, such as Northern Blot. One common problem in PCR is the non-specific amplifications of other gene products because cDNAs libraries of thousand of genes are often used as PCR templates. Therefore, we need to carefully design PCR primers that specifically amplify the genes of interest. Unfortunately, most available primer design programs only focus on primer chemical properties, such as melting temperature, GC content, secondary structure, etc. Little emphasis is given to primer mispriming to other genes. In contrast, all primers in PrimerBank were carefully designed to ensure gene specificity.

The primer selection algorithm has been extensively tested by real-time PCR experiments for PCR specificity and efficiency. Up to now, we have tested over a thousand primers and the design success rate is > 99%.

You may search PrimerBank by GenBank Accession, NCBI protein accession, LocusLink ID, or PrimerBank ID.

Search terms are automatically combined if they are separated by space. For example, search "kinase s6" returns all records with both keywords "kinase" AND "s6". AND and OR Boolean operators may also be used. Here are a few examples:

Currently, only a limited set of keywords (keywords defined in the protein definitions) can be searched. For example, you cannot search by gene symbols. If you did not find your genes, please look up the corresponding gene IDs (DNA accessions, proteins accessions, or LocusLink IDs) and search by IDs.

Because of the sequence redundancy in GenBank, each gene is usually represented by more than one NCBI record. PrimerBank sometimes uses the NCBI LocusLink index file to map multiple sequence records to the same gene locus. As a result, a different accession number other than originally submitted may be retrieved. However, both accessions represent the same gene. If a retrieved record has a different GenBank accession, a LocuLink ID will appear in the gene description field.