protocol_name: Cell culture media: McCoy's 5A medium containing 10% FBS, 4 mM glutamine, 10 u/ml penicillin, 10 ug/ml streptomycin
Extracted molecule
total RNA
Extraction protocol
protocol_name: RNA extraction with TRIZOL extraction_method: TRIZOL other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturer’s instructions. RNA integrity was assessed by the presence of 28S and 18S bands in denaturing agarose gel eletrophoresis and spectrophotometry with A260/A280 ratios > 2.0 after dilution in TE-buffer.
Label
cy3
Label protocol
protocol_name: Cy3 Label Protocol label_method: indirect labeling other: For the preparation of fluorescence-labeled cDNA targets, 20 µg total RNA for each channel (Cy3 and Cy5) were reverse-transcribed using Superscript II enzyme reaction kit (Invitrogen, Carlsbad, CA). Briefly, total RNA was mixed with 4 µg of oligo dT (20-mer) primer for a total volume of 25 µl, denatured at 70°C, and cooled to room temperature. Then, a mix containing 2.5 µl of 20 mM dNTP pool [10 mM of dATP, dGTP, dCTP, and 4 mM of dTTP (Amersham), and 6 mM of aminoallyl-dUTP (Sigma-Aldrich, St. Louis, MO)], 10 µl of 5X first strand buffer (Invitrogen), 5 µl of 0.1M dithiothreitol (Invitrogen), 1.0 µl of Superase Inhibitor (Ambion, Austin, TX), 3.2 µl of dH2O, and 3.3 µl of Superscript II reverse transcriptase were added and incubated for 60 minutes at 42°C, followed by a 5-minute incubation at 75°C. Remaining RNA was degraded by adding 2 µl of RNAse H (2 U/µl) (Invitrogen), followed by incubation at 37°C for 20 minutes. To remove traces of protein, 10 µl of Quick Clean Enzyme Removal Resin (Clontech, Palo Alto, CA) were added to the cDNA, mixed by 1-minute vortex, and centrifuged at 14,000 rpm for 1 minute. The resulting supernatant was filtered through a 0.65 µm Ultra-free MC filter (Millipore, Bedford, MA) to remove any resin residues. The cDNA probes were precipitated by adding 5.5 µl of 3M sodium acetate (pH = 5.2) and 137.5 µl of cold 190-proof ethanol, and incubated for 60 minutes at -20°C, followed by centrifugation for 15 minutes at 14,000 rpm and 4°C. The cDNA precipitate was washed twice with 70% ethanol, air-dried, and dissolved with 17 µl of 0.1M sodium bicarbonate. For the dye-coupling reaction, the monoreactive Cy3 (for control probes) and Cy5 dyes (for treated probes) (Amersham) were dissolved with 2 µl of dH2O, mixed with the corresponding cDNA probe and incubated for 60 minutes in the dark. The labeled cDNA samples were again precipitated by adding 2.5 µl of 3 M sodium acetate (pH = 5.2) and 50 µl of cold 190-proof ethanol, followed by an incubation for 90 minutes, and a centrifugation for 10 minutes at 14,000 rpm and 4°C. The supernatant was discarded, probes were washed twice with 70% ethanol, and air-dried. To remove unincorporated dye residues, probes were cleaned-up using the Nucleospin Extraction Kit (Clontech) following the manufacturer’s recommendations. To further clean-up the probes, the eluate from the previous step was washed twice with TE buffer (tris ethylenediaminetetraacetic acid) (pH = 7.4) using a Microcon YM-30 column (Millipore).
protocol_name: Cell culture media: McCoy's 5A medium containing 10% FBS, 4 mM glutamine, 10 u/ml penicillin, 10 ug/ml streptomycin
Extracted molecule
total RNA
Extraction protocol
protocol_name: RNA extraction with TRIZOL extraction_method: TRIZOL other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturer’s instructions. RNA integrity was assessed by the presence of 28S and 18S bands in denaturing agarose gel eletrophoresis and spectrophotometry with A260/A280 ratios > 2.0 after dilution in TE-buffer.
Label
cy5
Label protocol
protocol_name: Cy5 Label Protocol label_method: indirect labeling other: For the preparation of fluorescence-labeled cDNA targets, 20 µg total RNA for each channel (Cy3 and Cy5) were reverse-transcribed using Superscript II enzyme reaction kit (Invitrogen, Carlsbad, CA). Briefly, total RNA was mixed with 4 µg of oligo dT (20-mer) primer for a total volume of 25 µl, denatured at 70°C, and cooled to room temperature. Then, a mix containing 2.5 µl of 20 mM dNTP pool [10 mM of dATP, dGTP, dCTP, and 4 mM of dTTP (Amersham), and 6 mM of aminoallyl-dUTP (Sigma-Aldrich, St. Louis, MO)], 10 µl of 5X first strand buffer (Invitrogen), 5 µl of 0.1M dithiothreitol (Invitrogen), 1.0 µl of Superase Inhibitor (Ambion, Austin, TX), 3.2 µl of dH2O, and 3.3 µl of Superscript II reverse transcriptase were added and incubated for 60 minutes at 42°C, followed by a 5-minute incubation at 75°C. Remaining RNA was degraded by adding 2 µl of RNAse H (2 U/µl) (Invitrogen), followed by incubation at 37°C for 20 minutes. To remove traces of protein, 10 µl of Quick Clean Enzyme Removal Resin (Clontech, Palo Alto, CA) were added to the cDNA, mixed by 1-minute vortex, and centrifuged at 14,000 rpm for 1 minute. The resulting supernatant was filtered through a 0.65 µm Ultra-free MC filter (Millipore, Bedford, MA) to remove any resin residues. The cDNA probes were precipitated by adding 5.5 µl of 3M sodium acetate (pH = 5.2) and 137.5 µl of cold 190-proof ethanol, and incubated for 60 minutes at -20°C, followed by centrifugation for 15 minutes at 14,000 rpm and 4°C. The cDNA precipitate was washed twice with 70% ethanol, air-dried, and dissolved with 17 µl of 0.1M sodium bicarbonate. For the dye-coupling reaction, the monoreactive Cy3 (for control probes) and Cy5 dyes (for treated probes) (Amersham) were dissolved with 2 µl of dH2O, mixed with the corresponding cDNA probe and incubated for 60 minutes in the dark. The labeled cDNA samples were again precipitated by adding 2.5 µl of 3 M sodium acetate (pH = 5.2) and 50 µl of cold 190-proof ethanol, followed by an incubation for 90 minutes, and a centrifugation for 10 minutes at 14,000 rpm and 4°C. The supernatant was discarded, probes were washed twice with 70% ethanol, and air-dried. To remove unincorporated dye residues, probes were cleaned-up using the Nucleospin Extraction Kit (Clontech) following the manufacturer’s recommendations. To further clean-up the probes, the eluate from the previous step was washed twice with TE buffer (tris ethylenediaminetetraacetic acid) (pH = 7.4) using a Microcon YM-30 column (Millipore).
Hybridization protocol
protocol_name: Oligo Microarray Hybridization other: The probes were finally concentrated to a volume of 14 µl and mixed with 1 µl COT-1 DNA (8-10 µg/µl) (Invitrogen), 1 µl yeast tRNA (4 µg/µl) (Sigma), and 1 µl poly-dA DNA (8-10 µg/µl) (Amersham). Before hybridization, the probes were denatured for 2 minutes at 100?C and cooled to room temperature. We used 70-mer oligo glass slides featuring 22,149 spots, corresponding to approximately 9,800 unique Unigene Clusters (Qiagen Human Version 2.0 set), which were printed by the National Cancer Institute Microarray Facility, Advanced Technology Center, Gaithersburg, MD. Arrays were pre-hybridized for 60 minutes at 42°C using a pre-hybridization buffer consisting of 5X SSC (sodium chloride-sodium citrate), 1% bovine serum albumin, and 0.1% SDS (sodium dodecyl sulfate). They were then washed in dH2O, immersed in isopropanol for 2 minutes, and air-dried. Denatured probes were mixed at a 1:1 ratio with hybridization buffer consisting of 50% formamide (Ambion), 1X SSC, and 0.1% SDS, and hybridized onto the microarrays at 42°C for 14–16 hours in a water bath. After hybridization, the slides were washed for 1 minute in 1X SSC with 0.1% SDS, for 1 minute in 1X SSC, and twice for 1 minute in 0.2X SSC. The arrays were finally scanned at a 10µm resolution with a GenePix 4000A scanner (Axon Instruments, Foster City, CA) with variable voltages to achieve saturated signal intensities for about 1% of the total spot number.
Description
mAdb experiment ID: 48449
Data processing
After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table.
