Quantitative PCR for Gene Expression

qPCR was performed as previously described [21 (link)]. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to manufacturer's protocol. Contaminating genomic DNA was eliminated using RNase-free DNase (Qiagen). Total RNA (2μg) was reverse transcribed into single-stranded cDNA using the AMV First Strand cDNA synthesis kit (Roche) according to the manufacturer's instructions. Real-time PCR amplification reactions were performed using the SYBR Green PCR Master Mix (Applied Biosystems). The cycling conditions and the primers used to amplify IRS-1, IRS-4, and 18S have been described previously [18 (link)].

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Sanmartín-Salinas P, & Guijarro L.G. (2018). Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer. Journal of Oncology, 2018, 3812581.

Publication 2018

RNeasy Mini Kit RNase-free DNase AMV First Strand cDNA synthesis kit SYBR Green PCR Master Mix

Cdna Dnase Genomic Irs 1 Irs 4 Primers Rnase Sybr green Synthesis

Corresponding Organization : Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of IRS-1 and IRS-4 genes

control variables

RNA isolation using RNeasy Mini Kit (Qiagen)
Removal of contaminating genomic DNA using RNase-free DNase (Qiagen)
Reverse transcription of total RNA (2μg) into single-stranded cDNA using AMV First Strand cDNA synthesis kit (Roche)
Real-time PCR amplification using SYBR Green PCR Master Mix (Applied Biosystems)
18S rRNA as a reference gene for normalization

controls

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

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