Gender: female Age: premenopausal Tissue: normal breast Disease: none
Biomaterial provider
Brigham and Women's Hospital
Treatment protocol
immortalized by hTERT in WIT medium
Growth protocol
in vitro cultured in WIT medium on Primaria plates
Extracted molecule
total RNA
Extraction protocol
The standard protocols followed were per Affymetrix’s instructions (Genechip Expression Analysis Technical Manual 701021 Rev 4). Briefly, total RNA was isolated from cultured cells using Trizol (Invitrogen, Carlsbad, CA) as per manufacturer’s instructions. The RNA pellet was resuspended in 100 ul of RNAse free water. The RNeasy Clean-up kit (Qiagen, Valencia, CA) was utilized per manufacturer’s instructions resulting in 30 ul total RNA sample. The total RNA concentration and 260/280 ratio was evaluated on a NanoDrop ND- 1000 UV-Vis Spectrophotomter (NanoDrop Technologies, Rockland, DE). Only samples with a 260/280 ratio greater than 1.9 were further processed. An aliquot of 1 ul from each of the samples was diluted to be within the dynamic range of the Agilent RNA 6000 Nano Labchip kit (Agilent, Palo Alto, CA), with a target of 100 ng. The Nano Labchip protocol was followed as per manufacturer’s instructions and was placed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) for evaluation. Samples with the highest concentration, only 2 distinct 18 S and 28 S peaks, and no evidence of degradation were further processed. To obtain 15 µg of total RNA for first strand cDNA synthesis, samples were sometimes combined and placed on a spinvacuum to obtain the necessary concentration (1.66 µg/µl) and volume (9µl). Six hundred units of SuperScript II reverse transcriptase were used for the first strand cDNA synthesis reaction. The second strand DNA synthesis, the clean-up of the double-stranded cDNA, the synthesis of the biotin-labeled cRNA, and the clean-up of the biotin-labeled cRNA were per Affymetrix instructions. The Genechip Sample Cleanup module was used for the clean-up steps of the double stranded cDNA and the biotin-labeled cRNA. Quantification of the cRNA was evaluated on the NanoDrop. Samples were used for hybridization only if 20 µg of cRNA were obtained in an individual sample or by combining samples.
Label
biotin
Label protocol
As per Affymetrix instructions
Hybridization protocol
Fragmentation, hybridization, washing, staining, and scanning were done according to the Affymetrix protocol. Briefly, reagent preparation included 12X MES stock (1.22 M MES, 0.89 M [Na+]) and 2X hybridization buffer (1X hybridization buffer: 100mMMES, 1M[Na+], 20mMEDTA, 0.01% Tween 20). The hybridization cocktail components and final concentrations consisted of fragmented cDNA (.05 µg/µl), control oligonucleotide B2 (50 pM), 20X eukaryotic hybridization controls bioB, bioC, bioD, and cre (1.5, 5, 25, 100 pM, respectively), herring sperm DNA(.1 mg/ml), acetylated BSA (.5 mg/ml), 1X hybridization buffer with a final volume of 300 µl. The GeneChip array was filled with 250 µl of the hybridization cocktail and hybridized for 16 hours, rotated at 60 RPM, and maintained at 45C. Reagents prepared for washing and staining included a nonstringent wash buffer (6X SSPE, .01% Tween 20), a stringent wash buffer (100mMMES, 0.1M[Na+], 0.01% Tween 20), and a 2X stain buffer (1X: 100 mM MES, 1 M [Na+], 0.05% Tween 20). The wash procedure was carried out in an Affymetrix Fluidics Station 400 controlled by Affymetrix Microarray Suite 5.0 software resident on a personal computer. The fluidics station first went through a priming step and subsequently did the washing and staining by a software protocol.
Scan protocol
The microarrays were scanned using the GeneArray scanner per manufacturer’s instructions (Affymetrix, Santa Clara, CA). The quality control algorithms for eliminating an array are based on recommendations in both the Affymetrix and dChip software packages.
Description
Normal human mammary epithelial cells, hTERT-immortalized
Data processing
Affymetrix Microarray Suite 5.0. Microarray Suite 5.0
was used to generate a cell intensity file (*.cel). Probe intensities were
normalized and summarized using GCRMA (with default settings)
with R/Bioconductor.
