Spectra 2 plate reader

A human mimic microRNA library consisting of 2080 unique siRNA corresponding to known human microRNAs (MiRIDIAN, ThermoFisher) was used for screening microRNA that interact with the 3′-UTR of human CXCL8 mRNA. Immortalized colon fibroblasts (CT5 SV40) were seeded at 8000 cells per well in 96-well plates the day before transfection. These cells were immortalized via lentiviral transduction of hTERT SV40 (kind gift from Rosa Hwang, MD, MD Anderson). The next day each well of cells was co-transfected with unique library microRNA mimics (50 nM/well) and pMIRGLO-CXCL8-3′UTR plasmid using Dharmafect Duo (GE Dharmacon) according to the manufacturer protocol. After 48 hours luminescence was measured using DualGlo Luciferase Assay System (Promega) with a Spectra II plate reader (Molecular Devices). A non-targeting negative control and a positive control in form of a siRNA targeting the 3′UTR of human CXCL8 mRNA (5’-AUU CUA GCA AAC CCA UUC AUU-3′, Dharmacon) were included for each individual mimic 96-well plate screen.

Human colon fibroblasts were seeded in a 96-well plate at a concentration of 1 × 10⁴ cells per well. Twenty-four hours after seeding, fibroblasts were transfected with either parental pMIRGLO plasmid, wild type pMIRGLO-CXCL8-3′UTR or mutagenized pMIRGLO-CXCL8-3′UTR(mut) plasmid using Lipofectamine 2000. After 48 hours, luciferase activity was measured with DualGlo (Promega) and normalized to Renilla activity using a Spectra II plate reader (Molecular Devices).