Quantitative Analysis of Colonic Gene Expression

To assess gene expression in mouse colons, total RNA was isolated by pestle homogenization of 2 cm of tissue in TRIzol according manufacturer’s instruction. In rIEC6 cells, total RNA was isolated after treatment with 1 μg/mL LPS (Sigma) for the indicated times. The iScript cDNA synthesis kit (Bio-Rad, 170-8891) was used to synthesize cDNAs, in which gene expression was measured by quantitative PCR using the indicated primers (Table S2) and the SensiFAST kit (Bioline, Bio-96020) as described previously^{59 (link)}. The data is presented as relative expression (2^−ΔΔCt) with β-actin serving to normalize expression values.

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Eshelman M.A., Matthews S.M., Schleicher E.M., Fleeman R.M., Kawasawa Y.I., Stumpo D.J., Blackshear P.J., Koltun W.A., Ishmael F.T, & Yochum G.S. (2019). Tristetraprolin targets Nos2 expression in the colonic epithelium. Scientific Reports, 9, 14413.

Publication 2019

LPS iScript cDNA synthesis kit SensiFAST kit

Actin After treatment Cdnas Cells Colons Gene expression Mouse Primers Synthesis Tissue Trizol

Corresponding Organization :

Other organizations : Pennsylvania State University, National Institute of Environmental Health Sciences, Penn State Milton S. Hershey Medical Center, Mount Nittany Medical Center

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Variable analysis

independent variables

Treatment of rIEC6 cells with 1 μg/mL LPS (Sigma) for the indicated times

dependent variables

Gene expression measured by quantitative PCR using the indicated primers (Table S2) and the SensiFAST kit (Bioline, Bio-96020)

control variables

Pestle homogenization of 2 cm of mouse colon tissue in TRIzol according to manufacturer's instructions
Use of the iScript cDNA synthesis kit (Bio-Rad, 170-8891) to synthesize cDNAs
Normalization of gene expression values to β-actin

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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