Description

This track provides automatically-designed RT-qPCR primers for measuring the abundance of human and mouse transcripts using SYBR-based qPCR (qPCR with double-stranded DNA-binding reporter dye). The primers were generated by a procedure that targets all transcripts and all "possible" exon-exon and intron-exon junctions in the human and mouse transcriptomes.

Not all consecutive exon-intron-exon triplets generate "possible" primer pairs. "Possible" primer pairs are defined as satisfying a set of imposed design rules:

1. The first exon-exon junction is not addressed, to avoid problems related to abortive transcription.
2. Intron length should be more than 800 bp to avoid problems of double products in amplification.
3. Only junction primers are designed: forward and reverse primers must flank the junction.
4. Melting temperature of the primers should be between 60°C and 63°C (optimally 60.5°C, according to Breslauer et al., 1986).
5. Primer length should be 18-25 bp.
6. Product size should be 60-125 bp.
7. Primers are designed first for the intron-exon (pre-mRNA) junctions, and the two best primer pairs for each junction are chosen.
8. Then, for the reverse primer of each pair, two options are designed for the corresponding forward primer of the exon-exon (mRNA) junction.
9. The "Primer Mispriming Library" of the primer3 software is used: "human" for the human transcriptome and "rodent" for the mouse transcriptome.

The track provides easy access to primers for almost all transcripts in the transcriptome, eliminating the need for a tedious, error-prone design process.

Methods

The UCSC Genes model was used as a reference of the gene structure and the primer3 software as the design engine. The software goes over all possible exon-exon junctions in the transcriptome and applies our design rules/parameters to provide two primer pairs for every "possible" intron-exon junction and four pairs for every "possible" exon-exon junction.

Display Conventions

The primers to amplify pre-mRNA (intron-exon junctions) are shown in red and the primers to amplify mRNA (exon-exon junctions) in blue. For each pre-mRNA primer pair, there are two corresponding mRNA primers (that use the same reverse primer, if possible). Each pair has a unique code which stands for the gene name and the junction name. For example, the human pair "JAG1_uc002wnw.2_11_1" amplifies pre-mRNA, and the corresponding mRNA primers are "JAG1_uc002wnw.2_11_1_1" and "JAG1_uc002wnw.2_11_1_2."

Using JAG1_uc002wnw.2_11_1_2 to illustrate the naming scheme:

• JAG1 is the gene symbol.
• uc002wnw.2 is the UCSC Genes identifier of the isoform.
• 11 identifies the exon-intron-exon triplet.
• 1 is the number (1 or 2) of the intron-exon junction (step 7 in the Description section above). The names of primer pairs that cover intron-exon junctions end here. For the exon-exon junctions that use the same reverse primer, there is one additional number.
• 2 is the number (1 or 2) of the exon-exon junction pair (step 8 in the Description section above).

Clicking on a primer pair will take you to a new page with details for that pair. Additional properties for the primer pair, including forward and reverse sequence, melting temperature, GC%, and product size, are available by clicking on the number next to the instruction "Click here for primer details." There is also a batch query website available to download details for a large number of primers.

References

Breslauer KJ, Frank R, Blöcker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746-50. PMID: 3459152; PMC: PMC323600

Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365-86. PMID: 10547847

Zeisel A, Yitzhaky A, Bossel Ben-Moshe N, Domany E. An accessible database for mouse and human whole transcriptome qPCR primers. Bioinformatics. 2013 May 15;29(10):1355-6. PMID: 23539303