Multiplex Assays for Natriuretic Peptide Genes

Customized GeXP multiplex assays were designed to genes that included the natriuretic peptide system (see Supplementary Table S2). Target-specific reverse transcription (using 100 ng RNA as template), and PCR amplification were performed as we described previously [49 (link),55 (link),56 (link)], and in accordance with manufacturer’s instructions (Beckman Coulter, High Wycombe, UK). In brief, a master mix was prepared for reverse transcription as detailed in the GeXP starter kit (Beckman Coulter, High Wycombe, UK) and performed using a G-storm GS1 thermal cycler (GStorm Ltd., Somerset, UK), using the following protocol: 48 °C 1 min; 42 °C 60 min; and 95 °C 5 min. From this an aliquot of each reverse transcriptase reaction was added to PCR master mix, consisting of GenomeLab kit PCR reaction mix and Thermoscientific Thermo-Start Taq DNA polymerase. PCR reactions were performed using G-storm GS1 thermal cycler with a 95 °C activation step for 10 min, followed by 35 cycles of 94 °C 30 s; 55 °C 30 s; 70 °C 60 s. Products were separated and quantified using CEQTM 8000 Genetic Analysis System, and GenomeLab Fragment Analysis software (eXpress Analysis Version 1.0.25, Beckman Coulter, High Wycombe, UK).