Page 52 - 2022-23 BIOFACT Catalogue

Real-Time PCR Work flow

• Choose from singleplex, multiplex, low-throughput and high-throughput
Chemistry Selection qPCR depending on the type of analysis and decide according to purpose
(DNA binding Dyes whether to use DNA binding dyes, uorescent primer or probe type de-
or Probe type) pending on the cost of qPCR ampli cation, etc.
• In case of low-throughput singleplex qPCR, use DNA-binding dye such as
SYBR® Green I or EvaGreen™ Dye and in case of high-throughput qPCR, use
the uorescent primer or a probe.

• It is recommended to design the amplicon to be between 75 and 200bp.
• It is recommended to avoid the primer set that results in secondary structure or
Primer and binds to a long repeat single base (>4).
Probe Design • Ampli cation e ciency may be higher in case amplicon size is smaller.

• Annealing temperature should be set to 50 - 65℃ for qPCR ampli cation.
• It could be automatically designed when using the Primer 3
(Web-based primer design program, http://frodo.wi.mit.edu/applications/mfold/).

• In case speci city needs to be increased: Increase the annealing temperature (In-
hibits non-speci c ampli cation or primer dimer formation).
qPCR Optimization • Test the reproducibility of the ampli cation e ciency and the uorescence dy-
namic range of the primer designed through serial dilution of the template with
known concentration.
• Analysis e ciency should be 90 - 105% and R2 value from the standard curve
should be >0.980 or higher.
design program, http://frodo.wi.mit.edu/applications/mfold/).

• In case intercalating dye was used: Through checking not only the ampli cation
curve but also the melting curve, check whether there are any non-speci cally
Data Analysis ampli ed bands except the target gene.
(For Graph)
• Compare the Ct values between samples after checking that R2 of the standard
curve is close to 1 (maximum 0.99).

52 BIOFACT CATALOGUE