Sybr green qpcr mix

Total RNA was extracted from JEG-3 cells using the RNA-iSo PluS kit (cat. no. 9109; Takara Biotechnology Co., Ltd.). RNA was reverse-transcribed using cDNA Synthesis SuperMix (Shanghai Yeasen Biotechnology Co., Ltd.; cat. no. 11119ES60). RT was performed as follows: Initial hold at 25°C for 5 min, followed by 42°C for 30 min and final elongation at 85°C for 5 min. Synthesized cDNA was subjected to qPCR amplification using SYBR Green qPCR Mix (MedChemExpress; cat. no. HY-K0501A) and analyzed on ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.). Each 20 µl qPCR reaction mixture included 1.0 cDNA, 10.0 SYBR Premix Ex Taq (2X) and 0.4 µl each primer (10 µM) and was completed with double-distilled water. Amplifications followed a two-step cycling protocol as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 10 sec at 95°C and 30 sec at 60°C. The final step comprised the melting curve analysis. To normalize data, SFRP2 expression was compared with housekeeping gene GAPDH using the 2^−ΔΔCq method (27 (link)). The specific primer sequences were as follows: GAPDH forward, 5′-CCAGGTGGTCTCCTCTGA-3′ and reverse, 5′-GCTGTAGCCAAATTCGTTG-3′ and SFRP2 forward, 5′-CACCGAGGAAGCTCCAAAG-3′ and reverse, 5′-CTTTCGGACACACCGTTCAG-3′.

Total cellular RNA was extracted using Trizol (15,596,026, Thermo, USA). The cDNA reverse transcription kit (4,368,814, Invitrogen, USA) was used to convert RNA into complementary DNAs (cDNAs). Gene expression analysis was performed on the ABI 7900 system using SYBR Green qPCR Mix (HY-K0501A, MedChemExpress, USA). Using β-actin or U6 as an internal reference, gene level was calculated by 2^−ΔΔCt method. The primers sequences used were as follows:

miR-140-5p (F): GGGCCAGTGGTTTTACCCTA

miR-140-5p (R): GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCA

HDAC3 (F): CATCCAGATGTCAGCACCCG

HDAC3 (R): AAAGTAGGCTGAAGTCCCTGC

PTEN (F): CGACGGGAAGACAAGTTCAT

PTEN (R): AGGTTTCCTCTGGTCCTGGT

β-actin (F): CCCTGGAGAAGAGCTACGAG

β-actin (R): CGTACAGGTCTTTGCGGATG

U6 (F): CTCGCTTCGGCAGCACA

U6 (R): AACGCTTCACGAATTTGCGT