Gexp starter kit

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).

The reaction volume was 20 μL (containing 4 μL of 5 × RT buffers, 5 μL KanR RNA, 2 μL reverse primer, 1 μL reverse transcriptase enzyme (from GeXP Starter kit, Beckman Coulter, USA) and 5 μL RNA). In addition to the primer and Taq enzyme, other reagents were supported by a GenomeLab GeXP Start Kit (Beckman Coulter). The RT reaction was done in a thermal cycler (VWR-Doppio) under the following conditions: 1 min at 48 °C, 60 min at 42 °C, 5 min at 95 °C, and hold at 4 °C. Each experiment included a RT-Minus control and a no-template control (NTC) to ensure each peak provided the expected result. The RT-Minus control was a no-enzyme reaction to ascertain if the RNA was contaminated with DNA. The NTC was a no-template control to confirm all that reaction reagents were in good condition. The concentrations of all chemicals were not supported by the producer.

Customised GeXP multiplex assays were designed to genes that included the natriuretic peptide system as well as somatotrope markers (see Supplementary Table S1). Target-specific reverse transcription (using 100 ng RNA as template), and PCR amplification were performed as we described previously [15 (link),43 (link),44 (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 (Agilegene Technologies Ltd., Somerton, 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 Thermo Scientific 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 (Beckman Coulter, High Wycombe, UK).