Quantitative Gene Expression Analysis

Mouse liver and pancreas tissues were homogenized and total RNA was extracted using RNA-STAT 60 reagent according to the manufacturer’s protocol. The purity and quantity of total RNA were measured with a Nanodrop spectrometer (ND-1000, Thermo Fisher Scientific, Wilmington, DE) using ND-1000 V3.8.1 software and cDNA was reverse transcribed from 1 ug RNA using a one-step cDNA synthesis reagent (QScript cDNA Supermix, QuantaBio, Beverly, MA). RT-PCR was performed on the CFX384™ Real-Time System (Biorad, Hercules, CA) using iTaq Universal probe Supermix and Taqman probes as described elsewhere (Wahlang et al. 2019c (link)). The relative mRNA expression was calculated using the comparative 2^−ΔΔCt method and normalized against GAPDH mRNA.

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Jin J., Wahlang B., Shi H., Hardesty J.E., Falkner K.C., Head K.Z., Srivastava S., Merchant M.L., Rai S.N., Cave M.C, & Prough R.A. (2020). Dioxin-like and non-dioxin-like PCBs differentially regulate the hepatic proteome and modify diet-induced nonalcoholic fatty liver disease severity. Medicinal chemistry research : an international journal for rapid communications on design and mechanisms of action of biologically active agents, 29, 1247-1263.

Publication 2020

Nanodrop spectrometer (ND-1000, QScript cDNA Supermix CFX384™ Real-Time System iTaq Universal probe Supermix

Cdna Gapdh Liver Mouse Mrna Pancreas Rt pcr Synthesis Tissues

Corresponding Organization :

Other organizations : University of Louisville

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Variable analysis

independent variables

Tissue type (mouse liver and pancreas)

dependent variables

Relative mRNA expression of target genes

control variables

GAPDH mRNA expression (used for normalization)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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