Hieff pcr master mix with dye

Total RNA was reverse-transcribed using a Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China) according to the manufacturer’s instructions. For PCR, 2 × Hieff™ PCR Master Mix (With Dye) (Yeasen, Shanghai, China) was used. The cDNA and gDNA PCR products were evaluated using 2% agarose gel electrophoresis. The qPCR was conducted using 2 × SYBR Green qPCR Master Mix (Bimake, Houston, TX, USA). GAPDH, β-actin and U1 were used as controls, and relative expression levels were calculated using the 2^−ΔΔCt formula. All primer sequences are listed in Supplementary Table S1.

Venous blood was collected in tubes containing ethylenediaminetetraacetic acid. DNA was extracted from whole blood using a TIANamp Genomic DNA Kit (TIANGEN, cat. no.: DP304). Polymerase chain reaction (PCR)-restriction fragment length polymorphism assays were used to determine SNAP-25 gene (GenBank Accession Number D21267) MnlI (rs3746544) polymorphisms. The oligonucleotide primers used to determine the MnlI polymorphisms within the SNAP-25 gene have been described previously (Yang et al., 2020 (link)). The primers used to amplify the SNAP-25 gene (Yang et al., 2020 (link)) were as follows: forward, 5-TTCTCCTCCAAATGCTGTCG-3 and reverse, 5-CCACCGAGGAGAGAAAATG-3. PCR was performed in a 30-μL volume with 100 ng DNA, 20 pmol of each primer, 15 μL of 2 × Hieff PCR Master Mix (With Dye) (Yeason, cat. no.: 10102ES03), and ddH₂O. Amplification was performed using an automated thermal cycler (Techne Flexigene, Cambridge, UK). PCR conditions were as follows: 5 min for initial denaturation at 95°C, 35 cycles at 95°C for 45 s for denaturation, 1 min at 58°C for annealing, and 1 min at 72°C for extension, followed by 7 min at 72°C for final extension.

The total RNA from paired BC tissues or cells was extracted using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized using the HiScript III RT SuperMix kit (Vazyme Biotech, Nanjing, China). qRT-PCR was conducted using the SYBR Green Master Mix (Yeasen, Shanghai, China). Primer sequences were designed and synthesized by RiboBio (Guangzhou, China). 18 S was used as an internal reference for circRNAs, and ACTB was used as an internal reference for genes. The relative expression of circRNAs was assessed using the threshold cycle (CT) values. The PCR assay was conducted according to manufacturer’s instructions using the 2×Hieff™ PCR Master Mix (with dye) (Yeasen). The PCR products were obtained using different primers were subjected to 2% agarose gel electrophoresis, and the gel was scanned using the Gel Doc XR + imager (Bio-Rad, Hercules, CA, USA).