To obtain transient pre-miR-148b (PM10264, Ambion) expression, cells were plated in 6-well plates at 30-50% confluency and transfected 24h later using RNAiFect (QIAGEN, Stanford, CA) reagent, according to manufacturer’s instructions, with 75 nM pre-miR-148b. Cells were tested for miR overexpression 48h later.
Growth protocol
MDA-MB-231 cells were from American Type Culture Collection and maintained in standard conditions: Dulbecco's Modified Eagle's Medium containing 10 mM Glutamax and 4.5 g/mLglucose (DMEM Glutamax™, GIBCO Invitrogen Life Technologies, Carlsbad, CA), supplemented with 10% heat-inactivated FCS (Seromed, GmbH), 1 mM sodium pyruvate, 25 mM HEPES pH 7.4 and 100 μg/mL gentamicin (all from GIBCO Invitrogen Life Technologies, Carlsbad, CA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from 106 cells by using Trizol® Reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA was quantified using the ND-1000 spectrophotometer (Nanodrop), while RNA integrity and content of microRNAs (%) in each samples were assessed by capillary electrophoresis using the RNA 6000 Nano LabChip using the Agilent Bioanalyzer 2100 (Agilent Technologies). Only total RNA samples with R.I.N. (RNA Integrity Number) values of 6, or higher, were used for microarray analysis.
Label
Cy3
Label protocol
Eight hundred nanograms of total RNA were labelled with “Agilent One-Color Microarray-Based Gene Expression protocol” (Agilent) according to the manufacturer’s instructions. The synthesized cDNA was transcribed into cRNA and labelled with cyanine 3-labelled nucleotide. Labelled cRNA was purified with RNeasy Mini columns (Qiagen). The quality of each cRNA sample was verified by total yield and specificity calculated with NanoDrop ND-1000 spectrophotometer measurement (NanoDrop Technologies).
Hybridization protocol
Samples were hybridized to Whole Human Genome Microarray 4 x 44K (G4112F, Agilent Technologies). 1.65 ug of Cy3 labelled aRNA were hybridized for 17 hours at 65ºC in a hybridization oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to manufacturer's instructions (One-Color Microarray-Based Gene Expression Analysis, Agilent Technologies). Arrays were washed with Agilent Gene Expression wash buffers and Stabilization and Drying Solution as suggest by the supplier.
Scan protocol
Slides were scanned on an Agilent microarray scanner (model G2565CA) at 100% and 10% sensitivity settings.
Description
Agilent Feature Extraction software version 10.5.1.1 was used for image analysis.
Data processing
Only spots with signal minus background flagged as positive and significant (flag=1) were used in the following analysis as “detected” spot while probes with flag equal to 0 were noted as “null”. Probes with less than 73% of detected spots across all arrays were removed from the analysis. Background corrected intensities of replicated spots on each array were averaged. Data were inter-array normalized with Quantile normalization (Bolstad BM et al., Bioinformatics (2003), 19(2):185-93) and then log2-transformed. Principal component analysis, cluster analysis and profile similarity searching were performed with tMev that is part of the TM4 Microarray Software Suite (Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, et al. Methods in Enzymology. 2006;411:134-93). Identification of differentially expressed genes was performed with one and two class Significance Analysis of Microarray (SAM) program (Tusher VG, Tibshirani R, Chu G. Proc Natl Acad Sci USA 2001;98:5116-121) with default settings. SAM uses a permutation-based multiple testing algorithm and identifies significant genes with variable false-discovery rates (FDR). This can be manually adjusted to include a reasonable number of candidate genes with acceptable and well defined error probabilities.
miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS and CSF1
