mouse or ewe either virgin, in cycle, in gestation or lactation
Extracted molecule
total RNA
Extraction protocol
100 to 150 mg of snap-frozen mammary tissue samples were homogenized in 1 ml TRIZOL® Reagent (Invitrogen) using an Ultra-turrax, and then incubated for 5 minutes at room temperature to allow the complete dissociation of nucleoprotein complexes. An 0.2 ml volume of chloroform was added and the samples were shaken vigorously for 15 seconds, incubated at room temperature for 2 to 3 minutes and then centrifuged at 10,000 g for 15 minutes at 4°C. The aqueous phase was transferred to a fresh tube and the total volume of samples was measured. After the addition of 3 volumes of absolute ethanol, the RNA solution was purified using the NucleoSpin RNA clean-up kit (Macherey-Nagel), according to the manufacturer's guidelines. The quantity and integrity of RNA were estimated with a NanoDrop spectrophotometer (NanoDrop Technologies) and Agilent 2100 bioanalyser (Agilent Technologies). Only samples with an RNA integrity number ≥6 were used for subsequent miRNA expression analyses.
Label
Cy3
Label protocol
Agilent’s miRNA Complete Labeling and Hyb Kit (p/n 5190-0456) was used to generate fluorescently-labeled miRNA with a sample input of 100 ng of total RNA. This method involves the ligation of one Cyanine 3-pCp molecule to the 3' end of an RNA molecule with greater than 90% efficiency. Work was done according to the manufacturer's instructions.
Hybridization protocol
Cy3-labeled RNA was added to a hybridization cocktail, heated to 99°C for 5 minutes, transfered to an ice water bath for 5 minutes, then added to the miRNA array to hybridize at 55°C for 20 hours. Arrays were washed according to manufacturer's directions.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for miRNA array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters for miRNA arrays to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were flagged. Intensity data were normalized with Rosetta Resolver (ver. 7.2), using the Agilent/miRNA-Intensity Profile Builder. This process starts from raw scan data and saves reporter and sequence-level intensity profiles. This pipeline implements summarization from feature data as described in the Agilent Feature Extraction Software (v9.5) Reference Guide, 4th ed, 2007, 237–239. Features’ signal values are summarized to their reporter as a gTotalProbeSignal sum, and reporters are summarized to their sequence as a sum. This pipeline is not group dependent: replicates are not normalized as a group, with all operations performed scan-by-scan. Following this step, the Agilent/miRNA-Intensity Experiment Builder was used to do the group-level normalization. This pipeline starts from intensity-profile reporter data and builds sequence-level intensity experiments. This pipeline uses group processing. This pipeline differs from Agilent/Intensity Experiment builder pipeline by addition of 75th percentile scaling operation in the Normalize Intensities step (Inter-chip Scaling plug-in). The Inter-chip scaling plug-in also includes the option Trim outlying values from baseline data. This option is disabled in this pipeline, and should remain disabled because it can introduce biases in scaled profiles. The bias arises because outlier trimming excludes datapoints with negative values, and Agilent/miRNA data profiles can include many datapoints with small negative values.
