Quantitative gene expression analysis

Total RNA was isolated with Trizol reagent, and cDNA was synthesized using the superscript III preamplification system (Invitrogen). Real-time PCR (qPCR) was performed using the CFX Connect Real-Time PCR system (Bio-Rad Laboratories, Foster City, CA, USA) as described previously [7 (link)]. FAM dye-labeled TaqMan MGB probes and PCR primers for target genes were purchased from Applied Biosystems for human GDF15 (Hs00171132_m1), NDRG1 (Hs00608387_m1), NDRG2 (Hs0104515_m1), NDRG3 (Hs00259237_m1), maspin (Hs00985283_m1), AMPKα1 (Hs01562315_m1), AMPKα2 (Hs00178903_m1), and β-actin (Hs01060665_g1). The mean cycle threshold (C_t) values for target genes were normalized against the β-actin control probe to calculate ΔC_t values. All reactions were performed in at least three independent experiments.

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Hou C.P., Tsui K.H., Chang K.S., Sung H.C., Hsu S.Y., Lin Y.H., Yang P.S., Chen C.L., Feng T.H, & Juang H.H. (2021). Caffeic acid phenethyl ester inhibits the growth of bladder carcinoma cells by upregulating growth differentiation factor 15. Biomedical Journal, 45(5), 763-775.

Publication 2021

superscript III preamplification system CFX Connect Real-Time PCR system TaqMan MGB probes

Actin Cdna Gdf15 Genes Human Maspin Ndrg1 Primers Real time pcr Trizol

Corresponding Organization : Chang Gung University

Other organizations : Taipei Medical University, Taipei Medical University-Shuang Ho Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression levels of target genes: GDF15, NDRG1, NDRG2, NDRG3, maspin, AMPKα1, AMPKα2

control variables

RNA isolation method: Trizol reagent
CDNA synthesis method: Superscript III preamplification system (Invitrogen)
QPCR instrument: CFX Connect Real-Time PCR system (Bio-Rad Laboratories)
Endogenous control gene: β-actin

controls

Positive control: Not specified
Negative control: Not specified

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