Mouse specific taqman gene primers

PVN, ARC, and central amygdala were isolated via microdissection utilizing the brain punch technique and immediately frozen. Frozen tissue was homogenized in QIAzol Lysis Reagent using a TissueLyser II, with total RNA extracted using RNAeasy Lipid Tissue Mini kits and QIACube automated processing (Qiagen; Germantown, MD, United States). RNA concentration was measured with a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific; Waltham, MA, United States). cDNA was synthesized from total RNA using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific; Waltham, MA, United States). Quantitative real-time polymerase chain reaction (qPCR) was performed on a QuantStudio 12K Flex system (Applied Biosystems; Foster City, CA, United States) using mouse specific Taqman gene primers (ThermoFisher Scientific; Waltham, MA, United States). The primers used were: interleukin 6 (IL6; mm00446190_m1), tumor necrosis factor α (TNFα; mm00443258_m1), interleukin 1β (IL1β; mm00434228_m1), and interleukin 10 (IL10mm01288386_m1). Each sample was measured in triplicate with cycle threshold (CT) values normalized to 18S ribosomal RNA (Rn18s, mm03928990_g1). Relative gene expression was determined using the 2^–∆∆CT method.

In a subset of aged mice treated with Ang-(1-7) versus saline (n = 4–6/group), the heart was flash frozen in liquid nitrogen and stored at −80 °C to examine RAS, sympathetic and inflammatory markers using quantitative real-time PCR. Frozen heart tissue (25 mg) was homogenized in QIAzol Lysis Reagent using a TissueLyser II, with total RNA extracted using RNAeasy Lipid Tissue Mini Kits and the automated processing QIAcube (Qiagen, Germantown, MD, USA). RNA concentration was measured with a NanoDrop spectrophotometer (ND-1000). cDNA was synthesized from total RNA using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed on a QuantStudio 12K Flex system (Applied Biosystems, Foster City, CA, USA) using mouse-specific Taqman gene primers (ThermoFisher Scientific). The primers used were: AT1R (Agtr1a, mm01957722_s1), Mas receptor (Mas1, mm00434823_s1), ACE (mm00802048_m1), ACE2 (mm01159006 m1), Th (mm00447557_m1), TNFα (Tnf, mm00443258_m1), IL6 (mm00446190_m1), and IL10 (mm01288386_m1). Each sample was measured in triplicate with cycle threshold (CT) values normalized to the 18S ribosomal RNA (Rn18s; mm03928990_g1) housekeeping gene. Relative gene expression was determined using 2^−∆∆Ct methods.