Biomedical Engineering Reference
In-Depth Information
Table 4.3
Criteria for quantitative real-time PCR primers
Items
Recommendations
Note
Amplicon size
50-150 bp
If necessary, up to
200 bp
Primer size
19-23 nucleotides
GC content
35-65%
Annealing temperature
58-60°C
Annealing
temperatures of
forward and reverse
primers should be
similar.
Secondary structures
Low self- or heterodimer
formation
No more than 4 bases
should be involved
in secondary
structure formation.
Binding location
Exon-exon junction
Exon-exon junction
binding primers
reduce signal from
genomic DNA
contamination
Splice variants
Multiple or specifi c splice
variants
Depending on the
research purposes
and design primer sequences with the aid of commercially and publicly
available software (Table 4.4). Parameters, such as the size of the target
PCR product, length of primer base pair, sequential order of primer nucle-
otides, and ratio of primer CG to AT content, are critical to determining
an annealing temperature and product specifi city, and further affect PCR
effi ciency (Dieffenbach
et al.
, 1993; Wu
et al.
, 1991). Each primer set should
be validated for PCR effi ciency before use by testing serial dilutions of
cDNA templates (SABiosciences, 2008). Theoretically, the amount of PCR
product signaled by fl uorescence intensity is expected to increase two-fold
after each cycle if the amplifi cation effi ciency is 100 %. However, the effi -
ciency is greatly dependent on binding affi nity of primers to target DNA,
availability of target DNA, and availability of synthesis materials, such as
dNTPs, during PCR cycles. The fl uorescence intensity of each serial dilu-
tion of DNA templates at threshold cycle (C
T
, see section 'Data analysis')
is measured and plotted against the quantities of serial-diluted DNA to
determine the effi ciency for a particular primer set (Fig. 4.2a). Generally,
the amplifi cation effi ciency between 90% and 110% is considered accept-
able for a quantitative gene expression analysis (Raymaekers
et al.
, 2009).
In addition, if double-stranded binding dyes are used (i.e. SYBR
®
Green I),
PCR products are commonly evaluated by a melt curve analysis to ensure
