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