Quantitative Analysis of Kidney Gene Expression

Total RNA was isolated from kidneys using Tri reagent (Molecular Research Center, Inc, Cincinnati, OH, USA) per manufacturer’s protocol. Single-strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) for two-step qRT-PCR. Quantitative PCR was performed using Taqman gene expression assays (TNF-α: Mm00443260_g1, fibronectin: Mm01256744_m1, VEGFA: Mm01281449_m1, EGFR: Mm00433023_m1; GAPDH: Mm99999915_g1; Life Technologies, Grand Island, NY, USA) using a Bio-Rad CFX96 Real-Time System. Data were analyzed using Bio-Rad CFX Manager Software version 2.0 and relative expression quantified using the 2^(-∆∆CT) equation after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously [18 (link), 22 (link), 25 (link), 26 (link)].

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Awad A.S., You H., Gao T., Gvritishvili A., Cooper T.K, & Tombran-Tink J. (2015). Delayed Treatment with a Small Pigment Epithelium Derived Factor (PEDF) Peptide Prevents the Progression of Diabetic Renal Injury. PLoS ONE, 10(7), e0133777.

Publication 2015

Tri reagent iScript cDNA Synthesis Kit GAPDH CFX96 Real-Time System CFX Manager Software version 2.0

Assays Cdna Egfr Fibronectin Gene expression Glyceraldehyde 3 phosphate dehydrogenase Kidneys Synthesis Tnf α

Corresponding Organization : Pennsylvania State University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of the following genes:
- TNF-α
- Fibronectin
- VEGFA
- EGFR

control variables

GAPDH expression as the reference gene for normalization

controls

Positive control: None mentioned
Negative control: None mentioned

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