mRNA profiling of trifluridine resistant colorectal cancer cell lines and parental cell lines

microRNA and mRNA profiling was conducted for parental cell lines and cell lines resistant to trifluridine in 3 colorectal cell lines (DLD-1, HCT-116, RKO). We hypothesized that a detailed comparison between miRNA and mRNA expression might reveal the mechanism for acquired resistant to trifluridine in colorectal cancer. mRNA (1 mg) was extracted from parental cell lines and cell lines resistant to trifluridine in 3 colorectal cancer cell lines (DLD-1, HCT-116, RKO) using miRNeasy mini kit. cDNA and Amino Allyl aRNA was synthesized by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyeDye (Cy5) Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc. The labeled samples were hybridized for 16 h at 37 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc. Fluorescent array images were acquired with 3D-Gene Scanner (Toray Industries Inc., Tokyo, Japan) and Images were quantified using Extraction (Toray Industries Inc., Tokyo, Japan). The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals. Expression ratios were analyzed using normalized signal intensity.