Quantifying PGC1α and GAPDH mRNA Expression

For the determination of the mRNA expression of PGC1α and GAPDH, total RNA was extracted from 20–30 mg muscle using procedures we have previously described (3 (link)). The isolated RNA was used to perform cDNA synthesis using the ABI High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific Inc., Waltham, MA), and real-time PCR was performed on an Applied Biosystems 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA) using predesigned TaqMan® gene expression assays (probe/primer sets) for PGC1α (assay Hs00173304_m1) and GAPDH (assay Hs02786624_g1) (Thermo Fisher Scientific Inc., Waltham, MA).

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Tran L., Kras K.A., Hoffman N., Ravichandran J., Dickinson J.M., D’Lugos A., Carroll C.C., Patel S.H., Mandarino L.J., Roust L, & Katsanos C.S. (2018). Lower Fasted State but Greater Change in Plasma Amino Acid-Induced Rise in Muscle Protein Synthesis in People with Obesity. Obesity (Silver Spring, Md.), 26(7), 1179-1187.

Publication 2018

ABI High Capacity cDNA Reverse Transcription kit Applied Biosystems 7900HT Fast Real-Time PCR System TaqMan® gene expression assays

Assay Cdna Gapdh Gene expression Mrna Muscle Pgc1α Primer Real time pcr Reverse transcription Synthesis

Corresponding Organization : Mayo Clinic in Arizona

Other organizations : Purdue University West Lafayette

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression of PGC1α
MRNA expression of GAPDH

control variables

Amount of muscle tissue used (20-30 mg)
RNA extraction procedure
CDNA synthesis using ABI High Capacity cDNA Reverse Transcription kit
Real-time PCR using Applied Biosystems 7900HT Fast Real-Time PCR System
TaqMan® gene expression assays (probe/primer sets) for PGC1α and GAPDH

controls

No positive or negative controls were explicitly mentioned

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