Gene Expression Analysis by qRT-PCR

Total cellular RNA was extracted using an RNA Extraction kit (Tiangen Biotech Co., Ltd.). First-strand cDNA was synthesized using a Reverse Transcription kit (Tiangen Biotech Co., Ltd.) and subjected to real-time PCR analysis. PCR was performed in triplicate with an ABI Step One Plus Real-Time PCR (Applied Biosystems; Thermo Fisher Scientific, Inc.) under the following conditions: 95°C for 2 min followed by 40 cycles at 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 10 min as previously described (16 (link),17 (link)). Sequences of the gene-specific primers (sense and antisense, respectively) were: 5′-TCTTCCAGCCTTCCTTCCT-3′ and 5′-AGCACTGTGTTGGCGTACAG-3′ for β-actin; and 5′-CACCGCAATGGCTCCTTT-3′ and 5′-CACCAACTCTAGCAGCACATC-3′ for TIPE2.