Abi q6 detection system

Total RNA was extracted from vascular tissues exhibiting VVs and the matched ANVs of 20 patients using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Quality and concentration of RNA was assessed using NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific, Inc.). Isolated RNA was reverse transcribed into cDNA using RevertAid™ Fist Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR Green PCR kit (Qiagen, Inc.) and detected on an ABI Q6 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follow: 95°C for 10 min followed by 45 cycles of 95°C for 15 sec, 60°C for 1 min; the melting conditions were 95°C for 10 sec, 60°C for 1 min, 95°C for 15 sec for one cycle. U6 was used as the internal control, and relative quantification and calculations were done using the 2^−ΔΔCq method (27 (link)). Primers were designed by Ying Biotech. The primer sequences are listed in Table II.

Total RNAs from CC cell lines were acquired using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the concentration and purity of the RNA were determined. RNA was reverse-transcribed into cDNA using a RevertAid Fist Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. QPCR was performed using the ABI Q6 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with 1.0 µl cDNA and a SYBR^® Green Real-Time PCR Master mix (Takara Biotechnology Co., Ltd.). The PCR amplification included initial denaturation at 95°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 70°C for 30 sec. Relative quantification and calculations were performed using the comparative quantitation cycle method (2^−ΔΔCq) (21 (link)). GAPDH was used as the internal reference. The primer sequences used were as follows: E-cadherin forward, 5′-CCATTCCCAATGAGGCTGGT-3′ and reverse, 5′-GGCTTTTCTGTGACATCCGC-3′; vimentin forward, 5′-ACATGGTGGAAACCGAGGAT-3′ and reverse, 5′-TCCATTTCCCGCATTTGGT-3′; Snail forward, 5′-ACATGGTGGAAACCGAGGAT-3′ and reverse, 5′-GGTGGTGGAAGGAATAACGC-3′; and GAPDH forward, 5′-CGCTCCACCTTCAAGTATGC-3′ and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.

Total RNA was isolated and extracted from cells and tissues using Trizol reagent (Invitrogen). Then, Reverse transcription of RNAs was performed with a Reverse Transcription Kit (Takara) based on the manufacturer's protocols. The ABI Q6 detection system (Applied Biosystems Inc.) was used to run qPCR. The 2−ΔΔCt method was employed for relative quantification of gene expression. The qPCR primers were listed in Table S1. The template of METTL14 and lncRNA was standardized by GAPDH.

Total RNA was isolated from the spinal cords of rats in the SCI and SCI+EA groups 21 days after SCI using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the RevertAid™ First-Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), as per the manufacturer's instructions. Gene-specific RT primers were used for each miRNA.
RT-qPCR was performed for 45 amplification cycles with the following conditions: 95°C for 10 sec, 60°C for 60 sec, and 95°C for 5 sec. FastStart Universal SYBR Green Master mix (Thermo Fisher Scientific, Inc.) was used to amplify cDNA using the ABI Q6 detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). All reactions were set up in triplicate. Relative quantification and calculations were assessed using the comparative threshold cycle method (2^−ΔΔCq) (44 (link)). All primers were from Yingbiotech. Primer sequences and are listed in Table I. GAPDH and U6 were used as the internal control for mRNA and miRNA, respectively.