Quantitative real-time PCR

Total RNA was prepared with the Trizol reagent (Invitrogen) according to the manufacturer's instructions. For miRNA reverse transcription, cDNA was synthesized using TaqManH MicroRNA Reverse Transcription Kit (ABI) with 100 ng total RNA. For mRNA reverse transcription, cDNA was synthesized using ReverTra AceH qPCR RT Kit (TOYOBO) with 1 mg total RNA. Real-time PCRs were performed using SYBRH Select Master Mix for CFX (Invitrogen). Relative quantification was achieved by normalization to the amount of GAPDH (for mRNAs) or snRNA U6 (for miRNAs). The 2^−ΔΔCt (ΔCt = CtmiR-543-CtU6) method for quantitation of gene expression was used to determine miR-543 relative expression levels. The ΔΔCt was calculated by subtracting the ΔCt of the reference sample (a normal clinical or mouse tissue sample was chosen for the normalization of clinical samples or samples in two mouse models, LoVo cell was chosen for the normalization of five CRC cell lines) from the ΔCt of each sample. The mean relative miR-543 expression (5.8) of all clinical tumor samples was chosen as the cut-off to classify a tumor sample was High or Low according to their miR-543 expression level. The primers used are shown in Supplementary Table S2.