TEBs, mature ducts and distal stroma regions of mammary glands 2-5 were microdissected from anesthetised 5-week-old beta actin-GFP reportert mice using the Leica MZFLIII fluorescence microscope
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Trizol reagent (Invitrogen) using the manufacturers protocol and further ethanol precipitated to remove salt contaminants. The RNA was quantified using the Nanodrop 1000 spectrophotometre and the integrity of the RNA was determined using the Agilent 2100 bioanalyser. (Agilent Technologies, Santa Clara, CA)
Label
Cy3-pCp
Label protocol
100ng total RNA was dephosphorylated using calf intesinal phosphatase for 30 mins at 37°C, denatured at 100°C between 5 to 10 minutes, followed by being ligated to Cy3-pCp using T4 RNA ligase.
Hybridization protocol
10x GE blocking buffer and 2x Hi-RPM hybridization buffer was added to Cy3-pCp labelled total RNA to a final volume of 45uL, before being incubated at 100°C for 5 minutes and later transferred to the ice water bath for 5 minutes according to the manufacturer's instructions. Following that the labelled miRNA samples were hybridized to the Agilent mouse microRNA microarray (G4472A) in the Agilent Surehyb chamber (G2530-60029) for 20 hours at 55°C. After hybridization, the microarray was washed with GE wash buffer 1 (Agilent) for 5 minutes at room temperature, followed by GE wash buffer 2 (Agilent) for 5 minutes at 37°C. Prepare the hybridization chamber disassembly according to the manufacturers instructions.
Scan protocol
Microarray slides were scanned on the microarray scanner (G2565BA) utilising a color scan setting for 8x15k array slides (Scan area 61x21.6mm, Scan resolution 5um, Select eXtended Dynamic range, Dye channel is set to Green and Green PMT Is set to XDR Hi 100% and XDR lo 5%).
Description
microRNA expression of TEBs
Data processing
The scanned images were analysed with Agilent Feature Extraction Software version 9.5.3 or higher specifying the default miRNA protocol in the FE Grid Template properties. To set the FE Project properties, use default settings as suggested by manufacturer in the input section, in the Other section, select the miRNA_QCM_Apr07 from the QC Metric Set drop-down list. Select miRNA-v1_95_May07 as the selected protocol for miRNA microarrays. GeneView miRNA data files were collated, and gTotalGeneSignal and gIsDetected columns were extracted for each miRNA. Due to the presence of many slightly negative expression values (ie in the range -8 to 0), data were transformed by adding an offset of 8.0 to all values, thresholding all remaining values < 1.0 to 1.0, and then transformed by log-base-2. Data were quantile normalised, and restricted to human only miRNA’s, that were expressed in at least one sample, derived form the gIsDetected value, where 1 means detected, 0 means not detected on the microarray.
