Primer parameters defining efficiency and coefficient of determination in quantitative polymerase chain reaction

We performed a correlation analysis between primer parameters and qPCR efficiency/coefficient of determination in two independent samples from in vitro functional experiments.

Primer parameters do not define qPCR efficiency and coefficient of determination significantly if primers are designed according to the optimised PRIMER-BLAST settings.

Aim. To find the correlation between the primer parameters, efficiency, and coefficient of determination (R2 ) in quantitative polymerase chain reaction (qPCR) conditions.

Methods. Upon RNA isolation from primary human coronary artery endothelial cells, we performed reverse transcription-qPCR (RT-qPCR) utilising SYBR Green chemistry to measure the expression of the following genes: IL1B, IL6, CXCL8, IL12A, IL23A, PECAM1, VWF, KDR, FAPA, ACTA2, SMTN, VIM, COL4A1, MMP2, SNAI2, TWIST1, ZEB1, SCARF1, CD36, LDLR, VLDLR, VCAM1, ICAM1, SELE, SELP, CDH5, IL1R1, IL1R2, TNFRSF1A, TNFRSF1B, NOS3, PXDN. Primers were designed employing Primer-BLAST software using optimised settings. For the correlation analysis, Spearman's rank correlation coefficient was applied (GraphPad Prism).

Results. Coefficient of determination correlated with the primer pair rating by Beacon Designer, amplicon melting temperature, and GC content in the reverse primer. Reaction efficiency did not correlate with the Beacon Designer rating, yet being associated with length and GC content of the reverse primer. Abovementioned correlation coefficients ranged from 0.4 to 0.5 or from -0.4 to -0.5 indicative of moderate positive or negative correlation. Other parameters did not affect reaction efficiency and coefficient of determination. Conclusion Primer parameters do not define qPCR efficiency and coefficient of determination significantly if primers are designed according to the optimised PRIMER-BLAST settings. 

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