Accutarget

The HT-29 cell line (Korean Cell Line Bank, Seoul, Korea) was maintained at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics in a humidified atmosphere of 5% CO₂.
Knockdown of a specific gene was achieved by 24-h transfection of siRNA or a non-targeting control (AccuTarget, Bioneer, Daejeon, Korea) into HT-29 cells. To assess inflammatory responses, the cell culture medium was replaced with medium containing LPS (1–2 μg/mL) at 24 h post-transfection. Cells were harvested at 3 h for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis and at 24 h for immunostaining after LPS treatment.
Methods for immunohistochemical staining, qRT-PCR, and colitis mouse models are provided in the Supporting methods, Supporting Information 1. All experiments using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Yonsei University Severance Hospital, Seoul, Korea (IACUC Approval No: 2013-0166) and all methods were performed in accordance with the relevant guidelines and regulations.

We used 2.29 μL of RNA solution from 1 mL urine and 1.46 μL of reverse transcriptase solution of the TaqMan MicroRNA RT Kit (ThermoFisher Scientific) to produce complementary DNA (cDNA). The obtained cDNA solution (4.5 μL) was used for quantitative PCR (qPCR), qPCR analysis was performed using each miRNA-specific TaqMan primer (0.5 μL) and TaqMan Universal PCR Master Mix (4.5 μL, ThermoFisher Scientific) with an MX3000P system (Agilent; Santa Clara, CA, USA) and CFX Connect (Bio-Rad; Hercules, CA, USA). Mimic miRNAs (AccuTarget; Bioneer; Daejeon, Republic of Korea) were used to draw standard curves, and the net level of miRNA was calculated by a numerical formula. For normalizations, the obtained net level of miRNA was divided by the value of uCRE or that of the uRNA.