3.6. Quantitative Real-Time PCR

Total RNA was extracted by a total RNA purification kit (GeneMark, New Taipei City, Taiwan). The RNA concentration was quantified using a NanoVue Plus spectrophotometer (GE Health Care Life Sciences, Chicago, IL, USA). Quantitative PCR (qPCR) was performed on a real-time PCR detection system and software (Applied Biosystems, Foster City, CA, USA). First-strand complementary DNA (cDNA) was generated by SuperScript III reverse transcriptase kit (Invitrogen). Quantification of mRNA expression for genes of interest was performed by qPCR reactions with an equal volume of cDNA, forward and reverse primers (10 µM), and Power SYBR Green Master Mix (Applied Biosystems). The sequence of the PCR primers was as follows: ACE2: forward 5′-GCTGCTCAGTCCACCATTGAG-3′, reverse 5′-GCTTCGTGGTTAAACTTGTCCAA-3′; TMPRSS2: forward 5′-AATCGGTGTGTTCGCCTCTAC-3′, reverse 5′-GCGGCTGTCACGATCC-3′; GAPDH: forward 5′-TCCTGGTATGACAACGAAT-3′, reverse 5′-GGTCTCTCTCTTCCTCTTG-3′ [30]. The copy number of each transcript was calculated as the relative copy number normalized by the GAPDH copy number.