Quality Assurance / Quality Control
Primer Quality Control (QC)
Any algorithm's primer design must also be experimentally validated for
high-performance with wet bench quality control protocols starting with two
major success criteria.
- First, a melt curve analysis must verify that a single gene-specific
product is produced. Following the melt curve, an agarose gel can also
be run to further verify a single product of the predicted size, based
on the amplicon design, without primer dimers or off-target
- Second, the amplification efficiency must be greater than 90 percent
for accurate and reliable results. If a real-time RT-PCR assay does not
meet all of the above requirements, then the quality control fails, and
the assay must be re-designed.
All real-time PCR assays must generate a single band of the correct size
for the results to accurately represent the expression of the queried gene.
Secondary products confound the analysis. If using SYBR Green-based
detection, you can tell if your real-time PCR assays are specific enough by
simply running the default melting program on your instrument immediately
after the completion of the cycling program. A single peak indicates a
single melting event, and therefore a single product.
Of the various methods of determining amplification efficiency, the most
rigorous and classical method examines the slope of a calibration curve, much
like those used to assess dynamic range. An assay
Primer Design Algorithm
Primer design algorithm is key to effective qPCR based gene expression
analysis. Designs must meet several important thermodynamic and sequence
criteria. Primers are designed such that they must detect every alternative
transcript and splicing variant of the queried gene so as not to miss any
||50-150 base pairs
||Composition of last 3 base pairs
||Avoid primer self or cross- annealing
stretches >4 base pairs
||BLAST against human genome build 37
||Primer sequences do not include SNP with
>0.01 Average Heterozygosity
To do so, all known entries in the public databases should be found and
aligned to reveal a common gene-specific region for primer design.
By controlling the GC content, primer length, and the
primer melting temperature range, each assay can use a standard set of
PCR cycling conditions.
Uniform cycling conditions, in turn, allow researchers
to scale up from a single assay, to multiple assays on an entire 96- or
even 384-well plate.
Single Nucleotide Polymorphism (SNP) analysis can
eliminate repetitive sequences so that any individual source of total
RNA may be analyzed with the same assay.
BLAST analysis further insures that the chosen primer
sequences are sufficiently different from the rest of the transcriptome
in the species of interest.
Stability at the 3'-end of the primers controls the
start position for the DNA polymerase, further enhancing specificity.
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