Single Gene qPCR Primer Set is a service to verify the primers of dsDNA binding dye type used for real-time PCR analysis according to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guideline *. Gene information to be analyzed will be provided as a primer set according to the MIQE guideline * and the target specificity and PCR efficiency will be verified and provided as a primer set of 200rxn. We also provide verification services for the retention primer sequence. It can be used directly in the experiment without any further verification, and you can derive ready-to-publish data. * The MIQE Guidelines [Bustin, S.A., et al. 2009. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55:4, 611-622]
Single Gene qPCR Primer Set is a service that provides a primer set after performing a verification test to check whether it meets the required condition according to the MIQE guideline *. It can be easily used for experiments without any further verification and can save time for primer optimization. (Table References)
*MIQE Guideline?
MIQE (Minimum Information for Publication of Quantitative Real-time PCR Experiments) Guideline is for evaluating the reliability and reproducibility of qPCR experiment providing step-by-step checklists. This service provides the experimental information to the customers for them to evaluate the validity of the results.
Table. Information required for qPCR validation according to the MIQE guideline
| Item to check |
| Specificity (gel, sequence, melt, or digest) |
| For dsDNA binding dyes, Cq of the NTC |
| Calibration curves with slope and y intercept |
| PCR efficiency calculated from slope |
| r2 of calibration curve |
| Linear dynamic range |
| Cq variation at LOD |
| Evidence for LOD |
The MIQE Guidelines [Bustin, S.A., et al. 2009. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55:4, 611-622]
Target-specific primer design using primer blast and our bioinformatics tool
Exclusion of self primer-dimer formation sequence
Identification of single peak in the dissociation curve
Identification of single band in Gel electrophoresis
Short amplicon size of 70 ~ 150 bp
Wide amplification range of copies being about 102 ~ 107
qPCR amplification efficiency of 90-110% in compliance with the MIQE Guideline
• Applicable to Quantitative Real-time PCR
Figure. A diagram showing dissociation curve, dynamic range, standard curve, and electrophoresis image of the human ACTB and its target.
* As Single Gene qPCR Primer Set is verified with ExiCycler™ 96 V3 and 2X GreenStar™ qPCR Master Mix (-ROX Dye)(K-6253), results may be different if other company products are used.
