Abi 7300 sequencing detection system

Total RNA was extracted using TRIzol reagent (Invitrogen, U.S.A.), and conversed to cDNA using Superscript III reverse transcriptase (Invitrogen, U.S.A.). Quantitative real-time PCR (qRT-PCR) was performed with qRT-PCR Kits (Invitrogen, U.S.A.) according to the manufacturer’s instructions and an ABI 7300 Sequencing Detection System (Applied Biosystems, Foster City, CA, U.S.A.). The amplification conditions were as follows: initial denaturation at 95°C for 10 min, followed by 35 cycles of 10 s at 95°C, 15 s at 60°C, and 10 s at 72°C. The relative expression level of miRNA and mRNA was normalized to U6 small nuclear RNA and β-actin, respectively. The following primers were used for analysis: MALAT1 forward: 5′-AGGTAAAGCTTGAGAAGAT-3′, reverse: 5′-GGAAGTAATTCAAGATCAA-3′; miR-125b forward: 5′-CCAGATAC TGCGTATGTGTG-3′, reverse: 5′-GTCACCTGATCCCATCTAAC-3′; VE-cadherin forward: 5′-ATCGGTTGTTCAATGCGTCC-3′, reverse: 5′-CCTTCAGGATTTGGT ACATGACA-3′; U6 forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACG CTTCACGAATTTGCGT-3′; β-actin forward: 5′-AAATCTGGCACCACACCTT C-3′, reverse: 5′- GGGGTGTTGAAGGTCTCAAA-3′.

Total RNA was extracted from the cells using TRIzol^® reagent (Thermo Fisher Scientific, Burlington, ON, Canada) following the manufacturer’s instructions, and cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Real-time PCR (RT-PCR) was performed using Fast SYBR Green PCR Master Mix (Applied Biosystems, Burlington, ON, Canada) and an ABI 7300 Sequencing Detection System (Applied Biosystems, Burlington, ON, Canada). The PCR amplification conditions were: initial denaturation at 95 °C for 10 min, followed by 35 cycles of 10 s at 95 °C, 15 s at 60 °C, and 10 s at 72 °C. The real-time PCR primers used in this study are listed in Table 1.

Total RNA was extracted from the cells using TRIzol® reagent, and cDNA was synthesized using M-MLV-RT and Oligo-dT primers (Promega, USA) in 20 μL RT buffer. Real-time PCR was performed using the SYBR Premix Ex Tag Kit (TaKaRa, Japan) in an ABI 7300 Sequencing Detection System (Applied Biosystems, Foster City, CA, USA). The PCR was run for 1 cycle, 95°C, 10 min; 35 cycles, 95°C for 10s, 60°C for15s; and 1 cycle, 72°C for 10 s. The sequences of the oligonucleotide primers are shown as Table 1.