Human vastus lateralis muscle biopsy taken at day 4 of hospitalization for an acute COPD exacerbation
Extracted molecule
total RNA
Extraction protocol
Total RNA from the vastus lateralis muscle was isolated using the Trizol method (Gibco BRL, Life Technologies, Merelbeke, Belgium). In brief, approximately 0.1 g of muscle tissue was homogenized using an Ultra-Turrax homogenizer (Janke & Kunkel, Germany) in 1 ml Trizol Reagent. The homogenized samples were incubated at room temperature for five minutes and 0.2 ml chloroform was added. After shaking for 15 seconds the samples were incubated at room temperature for 2 minutes. Samples were centrifuged at 12,000 x g for 15 minutes at 4 ºC and the upper aqueous phase was transferred to a fresh tube. 0.5 ml isopropyl alcohol was added to precipitate the RNA. Samples were incubated at room temperature for 10 minutes and centrifuged at 12,000 x g for 10 minutes at 4 ºC. RNA pellet was washed with 75% ethanol and centrifuged at 12,000 x g for 5 minutes at 4 ºC. RNA pellets were briefly dried and dissolved in H2O.
Label
Cy5
Label protocol
Biotin-labelled cRNA target is prepared by a linear amplification method. The poly(A)+ RNA subpopulation (within the total RNA population) is primed for reverse transcription by a DNA oligonucleotide containing the T7 RNA polymerase promoter 5′ to a d(T)24 sequence. After second-strand cDNA synthesis, the cDNA serves as the template for an in vitro transcription (IVT) reaction to produce the target cRNA. The IVT is performed in the presence of biotinylated nucleotides to label the target cRNA. This method produces approximately 1000-fold to 5000-fold linear amplification of the input poly(A)+ RNA.
Hybridization protocol
CodeLink Gene Expression System: Single-Assay Bioarray Hybridization and Detection Protocol overview Hybridisation is performed overnight in a temperature-controlled shaking incubator. 1. Fragment cRNA. In 25 μl total volume, add 10 μg of cRNA to 5 μl of 5× fragmentation buffer and incubate at 94 ºC for 20 minutes. 2. Prepare hybridization reaction mixture. Bring 10 μg of fragmented cRNA, 78 μl of hybridization buffer component A, and 130 μl of hybridization buffer component B to a final volume of 260 μl with water. Incubate at 90 ºC for 5 minutes and immediately chill on ice for 5–30 minutes. 3. Loading reaction mixtures into array chambers. Slowly inject 250 μl of hybridization reaction mixture into array input port and seal ports with sealing strips. 4. Hybridization. Set the shaker speed to 300 rpm and incubate slides for 18–24 h at 37 ºC, maintaining a consistent hybridization time for comparative experiments. 5. Post-hybridization wash. Remove the Flex Chamber using the hybridization removal tool to hold the bioarray in place while peeling the Flex Chamber off at a 60º angle. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75× TNT. Transfer the bioarray rack to the large reagent reservoir containing preheated 0.75× TNT, and incubate at 46 °C for exactly 1 h. Do not exceed 1 h incubation. 6. Detection with streptavidin-dye conjugate. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir into the small reagent reservoir and incubate bioarrays at ambient temperature for 30 minutes. Wash the bioarrays four times with 1× TNT (5 minutes each wash) in large reagent reservoirs. Rinse the bioarrays in 0.1× SSC/0.05% Tween™ for 30 seconds. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark.
Scan protocol
Bioarray scanning and analysis. Scan bioarrays and analyze with CodeLink Expression Analysis software. Scanning with GenePix™ 4000B is described in this booklet. For use of CodeLink Bioarrays with other scanners, visit the CodeLink website (www.amershambiosciences.com). In brief: For scanning with a GenePix Array Scanner, use the following steps. For information on use of alternative scanners with CodeLink bioarrays, visit the CodeLink website (www.amershambiosciences.com). 7.1 Turn the scanner on 15 minutes prior to use. 7.2 Open the cover to expose the slide holder. 7.3 Lift the latch of the slide holder and lift the upper clip. 7.4 Wearing latex gloves, load the bioarray into the tray with the label side down and closest to the front of the scanner. 7.5 Pull the clip on the left of the slide out and let the bioarray fall into place. Release the clip to put pressure against the bioarray. 7.6 Grab bioarray by the edges and move the bioarray toward you. 7.7 Lower upper clip and press down on the latch until it clicks. 7.8 Slide the cover to the right to cover the slide holder. 7.9 Open the GenePix software and select the following settings: expand setting file name: Expression_5um.gps: whole genome products Expression_10um.gps: 10K and 20K products wavelength: 635 nm PMT voltage: 600 V laser power: 100% pixel size: 5μm for Whole Genome products 10μm for 10K and 20K products focus position: 0 μm 7.10 In the Report tab, open the scanning script by clicking Scan CodeLink Slide. 7.11 Enter the bioarray serial number and click Next. The Experiment and Scan Information interface is displayed. 7.12 Type in the project name, experiment name, and sample name. The username is automatically captured. When opening this interface for the first time, a message box may ask whether to allow an ActiveX interaction to proceed. Click Yes. 7.13 Select a setting (.gps) file. The standard setting file is CodeLinkExpr.gps. If a settings file was previously selected, the name and path are displayed under Current Settings File. To select a new file, click Browse under Select New Settings File. The values for project name, experiment name, user name, and settings file that were entered for a previous bioarray are retained but may be changed. 7.14 In the Load and Scan Slide screen, the standard TIF file name for the current bioarray is displayed. If desired, the name may be modified, however, the slide number as the first part of the name cannot be altered. 7.15 Click Browse to select the image path or the location where the image files will be stored. If a Security Alert message box is displayed, click Yes. 7.16 Click Scan Slide. The Image tab will display, and the instrument will perform the scan. 7.17 When complete, the view will return to the Report tab. 7.18 Click Save Image to save the scanned image at the appropriate file location. 7.19 Slide the cover to the left and remove the bioarray. 7.20 To scan the next bioarray, click New Slide and enter the serial number for the next bioarray. The setting information previously entered will be retained. 7.21 Analyze the image from each bioarray using CodeLink Expression Analysis software.
Description
COPD patients (forced expiratory volume in the first second/forced vital capacity (FEV1/FVC) <70%) hospitalized for an acute exacerbation were compared to clinically stable COPD patients visiting the outpatient clinic (FEV1/FVC <70%, no hospitalizations for acute exacerbations within 1 year before testing, no participation in training programs). On day 4 of hospitalization, percutaneous Bergström needle biopsies were taken from the vastus lateralis muscle. All participants gave oral and written informed consent to participate in the present study. Total RNA from the vastus lateralis muscle was isolated using the Trizol method (Gibco BRL, Life Technologies, Merelbeke, Belgium). Microarray analysis was performed on CodeLink UniSet Human 20 K Bioarray (Amersham Biosciences, Diegem, Belgium) according to the manufacturer’s protocol at the VIB MicroArray Facility (MAF, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium). In brief, the quality of the total RNA was assessed with the Agilent Bio-analyser and Nanodrop followed by two rounds of cDNA synthesis. Biotin-labelled cRNA was synthesized by in vitro transcription and quality checked with the Nanodrop. 10 µg of biotin-labelled cRNA was fragmented and hybridized on the microarray slides and subsequently scanned with an Agilent Scanner. Gene expression was measured with the Codelink Expression Analysis software.
Data processing
All the data processing was done by CodeLink™ Bioarrays software using default parameters set by the manufacturer.
