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 combinted 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 RNeasy 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
FAM
Label protocol
cDNA for each miRNA of interest was synthesized from an input of five nanograms total RNA using the TaqMan® microRNA Reverse Transcription Reagents and specific reverse transcription primers (Life Technologies, Carlsbad, CA, USA). Real-time PCR with TaqMan® probes was performed on a Life Technologies ViiA™ 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) using the following conditions: 10 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 60°C for 1 min. All assays were performed in triplicates.
Hybridization protocol
n/a
Scan protocol
n/a
Data processing
CT values were determined using the SDS software (Life Technologies, Carlsbad, CA, USA) with automatic baseline and threshold settings. The data was loaded into the R statistical environment (v2.14.0) and pre-processed. Triplicate CT values were averaged and normalized to the geometric mean of hsa-let-7g, hsa-miR-191 and hsa-miR-335-3p, which were selected as endogenous controls. CT values greater than 36 were considered to be below the limit of detection. Fold changes were calculated as Xenograft-Cell Line. Matrix non-normalized contains the mean of triplicate raw CT values. Matrix normalized contains log2|2^-ΔCT| values; where ΔCT = CT_target - CT_endogenous_controls. Fold Change contains CT values of xenografts compared to CT values of cell lines (ΔCT_xenograft - ΔCT_cell_line).
