storage: formalin-fixed parafin-embeded tissue tumor type: sporadic breast tumor
Extracted molecule
total RNA
Extraction protocol
For each sample tumoral area was marked by pathologist, FFPE blocks were cut in 5x 30µm sections and tumor tissue was isolated by needle macrodissection for subsequent RNA extraction. Total RNA was extracted using miRNeasy FFPE kit (QIAGEN) according to manufacturer’s instructions, while tissue digestion step was performed using 20 mg/ul Proteinase K (Roche, Basel, Switzerland) for over-night incubation step at 55ºC. RNA quantity was assessed by NanoDrop Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Label
Hy3
Label protocol
300ng of total RNA were treated for with Calf Intestine Phosphatase and than labeled with Hy3 fluorescent dye according to manufacturers instructions.
Hybridization protocol
Denaturated Hy3 labeled RNA samples in 1x hybridization buffer were hybridized at 56ºC for 16h onto Exiqon miRNA microarray slides (v.11.0 – hsa, mmu & rno) containing 1940 capture probes in 4 replicates, including 830 human miRNAs annotated in miRBasev.11 database and 434 hsa- miRPlus probes. A set of 10 synthetic spike-in RNAs was added to total RNA sample prior to labeling and later used for quality control.
Scan protocol
Processed slides were scanned with Agilent Array scanner (Agilent Technologies, Santa Clara, CA, USA), with the laser set to 635nm, at Power 80 and PMT 70 setting, and a scan resolution of 10μm.
Data processing
Fluorescence intensities on scanned images were quantified using Feature Extraction software (Agilent Technologies, Santa Clara, CA, USA) using the modified Exiqon protocol. Average processed intensity values of the replicate spots were background subtracted using Normexp, and log2 transformed. Raw data were quantile normalized for inter-array variability using R/Bioconductor. Data was further processed to eliminate miRNAs with uniformly low expression (<6.5) or with low expression variation (VAR<0.1) across the experiments, retaining 444 miRNA genes (276 hsa-miR + 168 hsa-miRPlus).
