Array pro image analysis software

miRNA microarray analysis including separation, quality control, labeling, hybridization and scanning was performed by LC Sciences (Houston, TX, USA). The array contained 1090 human mature miRNA probes for all human mature miRNAs based on Sanger miRase Release 15.0 (The Wellcome Trust Sanger Institute, Hinton, UK). Hybridization was performed on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, Inc., Houston, TX, USA). Images were collected using a laser scanner (GenePix 4000B; Molecular Devices, LLC, Sunnyvale, CA, USA) and digitized using Array-Pro image analysis software (Media Cybernetics, Inc., Rockville, MD, USA). The data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). Student's t-test analysis was conducted to identify differentially expressed miRNAs between MDA-MB-231 and MCF-7 cells. The false discovery rate (FDR) was P<0.05 and P<0.01 and served as the cut-off criteria. The data were log2 transformed and median centered by Cluster 3.0 software (Informer Technologies Inc., Los Angeles, CA, USA) and then further analyzed with hierarchical clustering with average linkage.

Microarray assay was performed using a service provider (LC Sciences, China). Six micrograms of total RNA sample was 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a μParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, USA).[10 (link)11 (link)] On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide-coding segment complementary to target miRNA or other RNAs and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 6× SSPE buffer (0.90 mol/L NaCl, 60 mmol/L Na₂HPO4, 6 mmol/L EDTA, and pH 6.8) containing 25% formamide at 34°C. After RNA hybridization, tag-conjugating Cy3 dye was circulated through the microfluidic chip for dye staining. Fluorescence images were collected and digitized using Array-Pro image analysis software (Media Cybernetics, USA). Data were analyzed by first subtracting the background and then normalizing the signals using a locally weighted scatterplot smoothing (LOWESS) regression.[12 (link)]