Abi prism 8500 system

WAT and BAT samples were homogenized in 1 mL of TRIzol reagent using a power homogenizer (Ultra-Turrax T10 basic, IKA-Works, Wilmington, DE, USA) to extract total RNA. After ethanol precipitation, total RNA was dissolved in 20–50 μL ribonuclease-free water, then converted to cDNA with SuperScrip IV Reverse Transcriptase (ThermoFisher Scientific) for real-time PCR analysis on an ABI Prism 8500 System (Applied Biosystems, Foster City, CA, USA) using SYBR green master mix reagent and the primer pairs listed in Table 1. Gene expression was calculated by the delta-delta CT method using the control housekeeping gene 36B4, a ribosomal protein, for normalization.

For RNA isolation, subcutaneous and viseral white adipose tissues were collected from wild type, SR-B1 knockout, and CD36 knockout mice. RNA were isolated using TRIzol® reagent (ThermoFisher Scientific, Waltham, MA), and followed the procedure from the manufacturer. After the ethanol precipitation step, total RNA was dissolved in 30 μl RNase free water, re-amplified to aRNA, then converted to cDNA using Superscript II reverse transcriptase (ThermoFisher Scientific, Waltham, MA). Real-time PCR was performed with the cDNA prepared as above using an ABI Prism 8500 System using SYBR green master mix reagent (Applied Biosystems Inc., Foster City, CA). The relative mass of specific RNA was calculated by the comparative cycle of threshold detection method (23 (link)) using acidic ribosomal phosphoprotein, large, P0 (36B4 or Rplp0) as the reference gene. Genes examined included: Fabp4, Cd36, Abca1, Acsl1, Fatp1, Fatp4, Sr-b1. Supplemental Table 1 shows the primer sets used for each gene.

BAT, EPI and subcutaneous fat of inguinal and backside (SUB-Q) tissue samples were homogenized in 1 ml of TRIzol^® reagent (15596026, ThermoFisher Scientific) using a power homogenizer Ultra-Turrax^® T10 basic (IKA^®-Works, Wilmington, DE, USA) in order to extract total RNA. After the ethanol precipitation step, total RNA was dissolved in 20–50 μl ribonuclease-free water then converted to cDNA with SuperScript^® IV Reverse Transcriptase (ThermoFisher Scientific) for real-time PCR analysis. Real-time PCR was performed with the cDNA prepared as above using an ABI Prism 8500 System (Applied Biosystems) using SYBR green master mix reagent and the primer pairs used listed in Table 1. Gene expression was calculated by the delta-delta CT method using the control housekeeping gene 36B4 rRNA.