The shRNA-Ascl2/HT-29 cells, shRNA-Ctr/HT-29 cells, shRNA-Ascl2/LS174T cells and shRNA-Ctr/LS174T cells were maintained in McCoy’s 5A medium (Sigma, USA) containing 10% fetal bovine serum (FBS; HyClone, USA), and 0.4 mg/ml G418 for HT-29 transfectants or 0.2 mg/ml G418 for LS174T transfectants, at 37ºC and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using TRIzol (Invitrogen) and the miRNeasy mini kit (QIAGEN) according to the manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity were measured by using a Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies), and RNA integrity was determined by gel electrophoresis.
Label
Hy3
Label protocol
The miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3TM fluorescent label using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
Hybridization protocol
After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.16.0) (Exiqon) according to the array manual. The total 25 μL mixture from Hy3TM-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm.
Scan protocol
Scanned on an Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA). Scanned images were imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction.
Description
shRNA-Ctr/LS174T cells Stable shRNA-Ctr/EGFP-transfected LS174T cells, harvested after several passages.
Data processing
Replicated miRNAs were averaged, and miRNAs with intensities >50 in all samples were chosen for calculating the normalization factor. Expressed data were normalized using the Median normalization. After normalization, differentially expressed miRNAs were identified through Fold Change (>=2.0) filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
The lists of differentially expressed genes are linked to the GSE34926 record as supplementary files.
