Sybr assay 1 low rox

From January 2015 to December 2016, we collected a total of 66 frozen surgically resected CRC tissues with AJCC stage II/III at The First Affiliated Hospital of Zhengzhou University. Follow up was concluded five years after surgery. Detailed baseline data of CRC patients were displayed in Additional file 1: Table S1. Total RNA was isolated from CRC tissues using RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s instructions. RNA quality was evaluated using a NanoDrop One C (Waltham, MA, USA), and RNA integrity was assessed using agarose gel electrophoresis. An aliquot of 1 µg of total RNA was reverse-transcribed into complementary DNA (cDNA) according to the manufacturer’s protocol using the miRNA reverse transcription Kit (TaKaRa BIO, Japan). All cDNA samples were prepared for qRT-PCR. This project was approved by the Ethics Committee Board of The First Affiliated Hospital of Zhengzhou University. In the qRT-PCR analysis, the enrolled 6 genes in the model were detected. qRT-PCR was performed using SYBR Assay I Low ROX (Eurogentec, USA) and SYBR^® Green PCR Master Mix (Yeason, Shanghai, China). The expression value of the target genes was normalized to GAPDH, and then log2 transformed for subsequent analysis. The primer sequences of the included 6 genes and GAPDH were shown in Additional file 1: Table S2.

We collected a total of 103 frozen surgically resected HCC tissues with AJCC stages I–III at The First Affiliated Hospital of Zhengzhou University. Detailed baseline data of HCC patients are displayed in Table 1. Total RNA was isolated from HCC tissues using RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s instructions. RNA quality was evaluated using a NanoDrop One C (Waltham, MA, USA), and RNA integrity was assessed using agarose gel electrophoresis. An aliquot of 1 µg of total RNA was reverse-transcribed into complementary DNA (cDNA) according to the manufacturer’s protocol using the mRNA reverse transcription Kit (TaKaRa BIO, Shiga, Japan). All cDNA samples were prepared for qRT-PCR. This project was approved by the Ethics Committee Board of The First Affiliated Hospital of Zhengzhou University. In the qRT-PCR analysis, the enrolled six genes in the model were detected. qRT-PCR was performed using SYBR Assay I Low ROX (Eurogentec, USA) and SYBR^® Green PCR Master Mix (Yeason, Shanghai, China). The 2^−ΔΔCt method was used to calculate the relative levels of gene and mRNA expression, and then log₂ transformed for subsequent analysis. The primer sequences of the included six genes and GAPDH are shown in Table S4.

Quantitative real-time PCR (qRT-PCR) was performed using SYBR Assay I Low ROX (Eurogentec, USA) and SYBR^® Green PCR Master Mix (Yeason, Shanghai, China) to detect gene expression. The 2^-ΔΔCt method was used to calculate the relative levels of gene expression. The primers are listed in Supplementary Table S2. GAPDH was used as the endogenous control for normalization. qRT-PCR assays were performed in triplicate with the following conditions: (1) 95°C for 5 min and (2) 40 cycles of 95°C for 10 s and 60°C for 30 s. The relative expression of genes was calculated using the ΔCT (Ct mRNA-Ct GAPDH) method. The sequences of qRT-PCR primers are listed in Supplementary Table S2.