Quantitative Gene Expression Analysis

Total cellular RNA was extracted using Ribozol^™ RNA extraction reagent (Amresco, Cleveland, OH, USA). Total RNA was quantified using Thermo Scientific^™ NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Purity of RNA was estimated by measuring the absorbance ratios at 260/280 and 260/230. Integrity of RNA was estimated using gel electrophoresis and visualizing the ribosomal RNA fractions. cDNA was synthesized using RevertAid reverse transcriptase (Thermo Fisher Scientific). qPCR was carried out using DyNAmo ColourFlash SYBR Green qPCR kit (Thermo Fisher Scientific) and specific primers in a Bio-Rad CFX96 Touch^™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). actb, hprt1 and polr2a were used as reference genes for qPCR. The three aforementioned genes have been identified as the most stable endogenous reference genes in the adipose tissue.16 (link) Expression levels of the target genes (abca1, slc2a4 and adipoq) were normalized to the geometric mean of the expression levels of the three reference genes.

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Vincent V., Thakkar H., Aggarwal S., Mridha A.R., Ramakrishnan L, & Singh A. (2019). ATP-binding cassette transporter A1 (ABCA1) expression in adipose tissue and its modulation with insulin resistance in obesity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 12, 275-284.

Publication 2019

Ribozol™ RNA extraction reagent Thermo Scientific™ NanoDrop 2000 spectrophotometer RevertAid reverse transcriptase DyNAmo ColourFlash SYBR Green qPCR kit Bio-Rad CFX96 Touch™ Real-Time PCR Detection System

Abca1 Adipose tissue Cdna Cellular Electrophoresis Expression genes Genes Primers Reverse transcriptase Ribosomal rna Sybr green Touch

Corresponding Organization :

Other organizations : All India Institute of Medical Sciences

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of the target genes (abca1, slc2a4 and adipoq)

control variables

Actb, hprt1 and polr2a were used as reference genes for qPCR
The three aforementioned genes have been identified as the most stable endogenous reference genes in the adipose tissue

controls

Positive controls: None mentioned
Negative controls: None mentioned

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