Quantifying PLAU mRNA Expression by RT-qPCR

Total RNA was extracted from the cells using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Single-strand cDNA was synthesized from 1 μg of total RNA using the Prime-ScriptTM Reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time quantitative PCR (RT-qPCR) primers were purchased from the Beijing Genomics Institute. The primers were as follows: PLAU sense, 5′-TCACCACCAAAATGCTGTGT-3′, and antisense, 5′-CCAGCTCACAATTCCAGTCA-3′ (Xu et al., 2015 (link)). The qPCR was conducted on a 7300 Real-Time PCR system (Applied Biosystems, Life Technologies, Singapore). The following cycling conditions were applied: 95 °C for 5 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 30 s. For each sample, qPCR assays were conducted in triplicate in a 10 μl reaction volume. β-Actin served as an internal control to normalize the expression of PLAU. The 2^−ΔΔCt method was employed to calculate the relative expression of PLAU mRNA (Livak & Schmittgen, 2001 (link)).