Cells were cultured in RPMI-1640, supplemented with 10% FBS and 1X Penicillin/Streptomycin. Xenografts were grown by subcutaneous injection of two million trypsin-dissociated tumor cells into non-obese diabetic/severe combined immunodeficient mice.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from confluent cell lines or fresh-frozen xenograft tissues using Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, followed by Dnase I treatment (Life Technologies) and purification using the Qiagen RNeasey kit (Qiagen, Venlo, Netherlands). RNA quantity and quality was assessed using the Qubit Fluorometer (Life Technologies), NanoDrop 1000 (Nanodrop Technologies, Wilmington, DE, USA), and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
n/a
Label protocol
The samples were prepared for nCounter miRNA expression profiling according to the manufacturer’s recommendations (NanoString, Seattle, WA, USA) by the University Health Networks (UHN) Microarray Centre (Toronto, Ontario, Canada).
Hybridization protocol
The samples were prepared for nCounter miRNA expression profiling according to the manufacturer’s recommendations (NanoString, Seattle, WA, USA) by the University Health Networks (UHN) Microarray Centre (Toronto, Ontario, Canada).
Scan protocol
The samples were prepared for nCounter miRNA expression profiling according to the manufacturer’s recommendations (NanoString, Seattle, WA, USA) by the University Health Networks (UHN) Microarray Centre (Toronto, Ontario, Canada). For each sample, a scan of 600 fields-of-view (FOV) was imaged.
Description
color-coded tags
Data processing
Prior to data normalization, nCounter data imaging quality control (QC) metrics were assessed. There was no significant discrepancy between the FOVs attempted and the FOVs counted. The binding density for the samples ranged between 0.24 and 0.72 – within the typical recommended range. The raw data was loaded into the R statistical environment (v2.14.0), and re-annotated against miRBase v16. Prior to normalization, probes indicated to have some level of background were corrected using probe level adjustment factors. Then, the geometric mean of the positive controls was used for code count normalization, while the background was estimated using the mean of the negative controls. Sample input amounts were normalized to the geometric mean of five housekeeping mRNA controls (ACTB, B2M, GAPDH, RPL19 and RPL10) included in the assay, and finally, to total miRNA count.
