4.3. RNA Isolation, Conventional RT-PCR, and Quantitative Real-Time RT-PCR (qRT-PCR)

An RNeasy mini kit (Qiagen, Valencia, CA, USA) was used to extract total cellular RNA based on provide protocols. The All-in-One First-Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China) was then used for cDNA reverse transcription based on provided protocols. Study primers were: Human ESRP1 (HQP013766, GeneCopoeia); human cyclin A2 (HQP021701, GeneCopoeia, Inc.); human GAPDH (HQP064347, GeneCopoeia); human TK-1, 5′-TTAACCTGCCCACTGTGCTGCCT-3′ (sense) and 5′-TTCTTGAAGTAGCAGAGCCGACAC-3′ (antisense); human GAPDH, 5′-TGT CGT GGA GTC TAC TGG TG-3′ (sense) and 5′-GCA TTG CTG ACA ATC TTG AG-3′ (antisense); human CD44, 5′-GCACTTCAGGAGGTTACATC-3′ (sense) and 5′-ACTGCAATGCAAACTGCAAG-3′ (antisense). The All-in-One Qpcr Mix Kit (GeneCopoeia) was used for all qRT-PCR reactions, following provided directions and utilizing an ABI 7500 FAST platform (Applied Biosystems, Foster, CA, USA). Conventional PCR was performed with 2.5 U LA Taq polymerase (Takara, Dalian, China). Thermocycler settings were: 95 °C for 3 min, 35 cycles of 95 °C 30 s, 55 °C 20 s, 72 °C 1 min, and then 72 °C for 5 min.