Colon RNA Extraction and qPCR Analysis

Total RNA was isolated from the colon using a commercial kit (Nucleospin RNA/Protein; Macherey-Nagel, France). Proteins and mRNA are obtained from the same colon sample and not from two portions of the same sample using this kit. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was carried out in a final volume of 20 µL from 1 µg total RNA (high capacity cDNA RT kit; Applied Biosystems, Villebon Sur Yvette, France). cDNA was amplified by PCR using Fast SYBR green (Applied Biosystems) in the one-step system (Applied Biosystems). SYBR green dye intensity was analyzed using one-step software. All results were normalized to the reference gene, POLR2A [50 (link),51 (link)].

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Charlet R., Sendid B., Kaveri S.V., Poulain D., Bayry J, & Jawhara S. (2019). Intravenous Immunoglobulin Therapy Eliminates Candida albicans and Maintains Intestinal Homeostasis in a Murine Model of Dextran Sulfate Sodium-Induced Colitis. International Journal of Molecular Sciences, 20(6), 1473.

Publication 2019

Nucleospin RNA/Protein high capacity cDNA RT kit Fast SYBR green one-step system

Cdna Colon Fast green Gene Mrna Proteins Reverse transcription Spectrophotometry Sybr green

Corresponding Organization :

Other organizations : Institut de Biologie de Lille, Centre Hospitalier Universitaire de Lille, Université de Lille, Inserm, Lille Inflammation Research International Center, Centre de Recherche des Cordeliers, Sorbonne Paris Cité, Université Paris Cité, Sorbonne Université

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels

control variables

Total RNA isolated from the colon using a commercial kit (Nucleospin RNA/Protein; Macherey-Nagel, France)
RNA quantification performed by spectrophotometry (Nanodrop; Nyxor Biotech, France)
Reverse transcription of mRNA carried out in a final volume of 20 µL from 1 µg total RNA (high capacity cDNA RT kit; Applied Biosystems, Villebon Sur Yvette, France)
CDNA amplified by PCR using Fast SYBR green (Applied Biosystems) in the one-step system (Applied Biosystems)
SYBR green dye intensity analyzed using one-step software
All results normalized to the reference gene, POLR2A

controls

No positive or negative controls explicitly mentioned

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