|
| Status |
Public on Mar 28, 2012 |
| Title |
MDCK_pH7.4_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Renal tubule cells, pH 7.4, replicate 2
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: Madin Darby Canine Kidney cells (MDCK)
ph: Physiological
|
| Treatment protocol |
On the day of study, cells were divided and placed either in media with a pH of 7.4 or one with a pH of 7.0 produced by reducing media bicarbonate concentration. They were then returned to the incubator for periods of 24 hours. The pH of the media while residing in the incubator was confirmed by measuring pH with an in situ pH electrode.
|
| Growth protocol |
Madin Darby Canine Kidney cells (MDCK) were obtained from the American Type Culture Collection. Cells were grown to confluence in 5 ml of DMEM with fetal bovine serum (pH 7.3 to 7.4) for 3 to 5 days in an aerobic environment 20% O2, 5% CO2 and 75% N2 at 37ºC. Twenty-four hours prior to the experiment, culture media was switched to serum-free DMEM/Hams-F-12 pH 7.4.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the MDCK cells held at pH 7.4 or 7.0 for 24hrs using TRIzol reagent (Invitrogen, Carlsbad, California) combined with RNeasy columns (Qiagen, Valencia, California) and the Qiagen Eukaryotic RNA isolation kit. The cells were collected from the experimental flask and lysed in 1 ml of TRIzol reagent. To the extract the RNA from the lysed cells, 100mL of chloroform was added per 500ul of cell lysate. The cell extraction mixtures were repeatedly inverted in a microcentrifuge tube for 15 seconds, after which they were centrifuged at 12,000 rpm at 4ºC for 10 min. The clear supernatant was mixed with 250 mL of ethanol, transferred to the RNAeasy Spin column and RNA purification was performed following the manufacturer’s instructions. The RNA concentration was quantified by absorbance at 260nm, and the quality was evaluated by capillary electrophoresis using a model 2100 Bioanalyzer with a RNA 6000 Nano Assay kit (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1.0 ug RNA using the One-Color Microarray-Based Gene Expression Analysis Quick Amp Labeling protocol (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 (3.7.1) Spectrophotometer.
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| |
|
| Hybridization protocol |
To prepare the cRNA for hybridization the Agilent Fragmentation mix was used according to the Quick Amp Protocol. After a 30 minute incubation period at 60º C, 55uL of 2x GE Hybridization Buffer HI-RPM was added to each reaction tube. Samples were then vortexed and briefly spun for 1 minute at 13,000 RPM to collect the sample at the bottom of the tube. Samples were then loaded onto the microarray slides and incubated in a hybridization chamber for 17 hours at 65 º C. The preparation was then washed with Agilent’s Gene Expression Wash Buffer 1 and 2 (with Triton X) according to the protocol and scanned using the DNA Microarray Scanner11.0.2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 24H incubation in pH 7.4 media
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20101102) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data were filtered by expression (20-100%) using GeneSpring GX Software 11.5.1. All data with at least a two- fold change compared to control were subjected to a T-test. Only those genes whose expression was altered by at least 2 fold with a P-value less than 0.05 were considered to represent significantly regulated genes.
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| |
|
| Submission date |
Mar 27, 2012 |
| Last update date |
Mar 28, 2012 |
| Contact name |
Thomas Thong Nguyen |
| Organization name |
Sachs Lab
|
| Street address |
11301 Wilshire Blvd
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90073 |
| Country |
USA |
| |
|
| Platform ID |
GPL15379 |
| Series (1) |
| GSE36848 |
Acid Stress Increases Gene Expression of Proinflammatory Cytokines in Madin Darby Canine Kidney Cells |
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