RT-qPCR for Transcriptional Analysis

Purified mRNA was converted to cDNA using the High-Capacity RNA-to-cDNA kit (4387406, ThermoFisher). RT-qPCR was performed with pre-mixed Taqman primer/probes (4331182, ThermoFisher; see S3 Table) and Taqman master mix (4369016, ThermoFisher) using 4 ng cDNA per well (10 μL reactions). 384-well plates were robotically loaded using the Biomek 3000 (Beckman Coulter, Indianapolis, IN) and analyzed using the 7900HT Fast Real-Time PCR System (Fisher Scientific). Each gene of interest was normalized to the geometric mean of three housekeeping genes (18s, rplp0, and actb); the Ct values of these housekeeping genes did not differ between dietary conditions. Statistical tests and linear modeling were conducted using ΔΔCt values to omit floor effects (normalized to RC NoDS) and data are presented as percent change from RC NoDS [30 (link)].

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Perron I.J., Keenan B.T., Chellappa K., Lahens N.F., Yohn N.L., Shockley K.R., Pack A.I, & Veasey S.C. (2018). Dietary challenges differentially affect activity and sleep/wake behavior in mus musculus: Isolating independent associations with diet/energy balance and body weight. PLoS ONE, 13(5), e0196743.

Publication 2018

High-Capacity RNA-to-cDNA kit Taqman primer/probes Taqman master mix Biomek 3000

Cdna Dietary Gene Housekeeping genes Mrna Nods Primer Rplp0

Corresponding Organization : University of Pennsylvania

Other organizations : Translational Therapeutics (United States), National Institute of Environmental Health Sciences, National Institutes of Health

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Variable analysis

independent variables

Dietary conditions

dependent variables

Gene expression (mRNA levels)

control variables

Levels of housekeeping genes (18s, rplp0, and actb)
CDNA amount (4 ng per well)

controls

Positive control: Not specified
Negative control: Not specified

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