Quantitative RT-PCR Gene Expression Analysis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc., Redwood City, CA) with on-column DNase digestion using the RNase-Free DNase Set (Qiagen Inc., Redwood City, CA). Following reverse transcription into cDNA with the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Sciences, Indianapolis, IN), samples were analyzed by real time PCR on a 7500 Real Time PCR System instrument (Applied Biosystems, Inc., Foster City, CA) using EagleTaq Universal Master Mix (Roche Applied Sciences, Indianapolis, IN). Target gene expression was normalized to 18S rRNA expression. The primers and probes used in this study are displayed in S7 Table, or have previously been described [24 (link),62 (link)–65 (link)]. Note that, although there was insufficient mRNA from biopsy samples to perform extensive qRT-PCR validation of gene expression, in contrast to microarray, RNA-Seq has high concordance with qRT-PCR data [28 (link)].

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Fletcher S.P., Chin D.J., Gruenbaum L., Bitter H., Rasmussen E., Ravindran P., Swinney D.C., Birzele F., Schmucki R., Lorenz S.H., Kopetzki E., Carter J., Triyatni M., Thampi L.M., Yang J., AlDeghaither D., Murredu M.G., Cote P, & Menne S. (2015). Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B. PLoS Pathogens, 11(9), e1005103.

Publication 2015

RNeasy Mini Kit RNase-Free DNase Set Transcriptor First Strand cDNA Synthesis Kit 7500 Real Time PCR System instrument EagleTaq Universal Master Mix

18s rrna Biopsy Cdna Digestion Dnase Gene expression Microarray Mrna Primers Real time pcr Redwood Reverse transcription Rna seq Rnase Synthesis

Corresponding Organization : Georgetown University Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Target gene expression normalized to 18S rRNA expression

control variables

Total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc., Redwood City, CA) with on-column DNase digestion using the RNase-Free DNase Set (Qiagen Inc., Redwood City, CA)
Reverse transcription into cDNA with the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Sciences, Indianapolis, IN)
Real-time PCR analysis using EagleTaq Universal Master Mix (Roche Applied Sciences, Indianapolis, IN) on a 7500 Real Time PCR System instrument (Applied Biosystems, Inc., Foster City, CA)

controls

Positive control: None mentioned
Negative control: None mentioned

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