breed: Holstein cell: Control epithelial location: embedded layer epithelium
Treatment protocol
An optimized method for BrdU immunostaining that retains RNA quality in tissue cryosections was used (Choudhary et al., J Dairy Sci 93 (6):2574-2579) to identify the label retaining epithelial cells (LREC). Immediately after staining slides were examined and cells excised with a laser microdissection system equipped for epifluorescence microscopy (Leica AS-LMD, Mannheim, Germany). Four categories of LREC and contol (non-LREC) epithelial cells were excised. The four categories were LREC from basal (LRECb) and embedded epithelial layers (LRECe), and epithelial control cells from basal (ECb) and embedded layers (ECe).
Growth protocol
Mammary tissue was collected from the outer parenchymal region (region in close proximity to the border with mammary fat pad) of a rear mammary gland. Individual samples were immediately embedded in OCT compound (Sakura, Torrance, CA, USA), frozen in liquid nitrogen vapor and stored at -80°C until use.
Extracted molecule
total RNA
Extraction protocol
Cells within the cap were dissolved in 2 µl of lysis buffer (WT-Ovation™ One-Direct RNA Amplification System; NuGEN Technologies, Inc. San Carlos, CA). The tube was capped and centrifuged for one minute at 13000 rpm, after which the tube and contents were vortexed gently for 30 seconds and centrifuged briefly before placing on ice. First stand cDNA synthesis and amplification reaction was carried out using Ribo-SPIA-based RNA amplification according to the manufacturer’s recommendations. Concentrations of amplified cDNA were determined spectrophotometrically (ND-1000, NanoDrop Technologies, Rockland, DE). A known amount of RNA (250 pg, RIN 7.5) was used as positive control for cDNA amplification. Nuclease-free water was used as a no-template control of cDNA amplification.
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
Control cells (6-13 objects) laser-microdissected dissected from an embedded epithelial layer
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
