Multiplex Analysis of Natriuretic and Gonadotrope Genes in Mice

Tissue was collected from 5 to 8 C57/B6 male and female mice in accordance with UK Home Office Guidelines (PPL70/6965), placed into either a 1.5 mL Eppendorf tube or foil (brain and heart tissue) and snap frozen in liquid nitrogen. Total RNA was extracted from primary mouse tissue (cardiac ventricle, adrenal, adipose, brain, pituitary, kidney, liver and testis), or from cultured αT3-1 or LβT2 cells, using RNAbee reagent, and subjected to DNase treatment (Qiagen, Poole, UK), as described previously [8 (link)]. RNA concentrations were determined using ND-100 spectrophotometer (Nanodrop, Thermo Fisher, Hemel Hempstead, UK). Two customised GeXP multiplex assays were designed, to detect natriuretic peptide gene targets (Nppa, Nppb, Nppc, Npr1, Npr2, Npr3, Corin and Furin), or gonadotrope transcription factor genes (Nr5A1, Nr0B1, cJun, cFos and Egr1) (Table S1). In all assays, 100 ng of total RNA was used per sample. Target-specific reverse transcription and PCR amplification was performed as previously described [36 (link)] and in accordance with manufacturer’s instructions (Beckman Coulter, High Wycombe, UK). In brief, a master mix was prepared for reverse transcription reactions as detailed in the GeXP Starter Kit (AB Sciex, Warrington, Cheshire, UK), and performed using a G-Storm GS1 thermal cycler, using the programme protocol: 48 °C for 1 min, 42 °C for 60 min, and 95 °C for 5 min. From this, an aliquot of each reverse transcription reaction was added to PCR master mix containing GenomeLab kit PCR master mix (AB Sciex, Warrington, Cheshire, UK), and Thermoscientific Thermo-Start Taq DNA polymerase (Thermo Fisher; AB Sciex, Warrington, Cheshire, UK). PCR reaction was performed using a 95 °C activation step for 10 mins, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 secs and 70 °C for 60 secs. Products were separated and quantified using the GeXP CEQ^TM 8000 Genetic Analysis System AB Sciex, Warrington, Cheshire, UK), and GenomeLab Fragment Analysis software (eXpress Analysis Version 1.0.25, Beckman Coulter, Inc.).