Total DNA was extracted following conventional DNA extraction methods.
Label
Cy5
Label protocol
1.- DNA from each sample were primed with 3µl of random hexamers d(N)6 0.5mg/ml (Promega) at 70ºC for 10min. For each labeling reaction, a master mix was prepared: 5µl First Strand buffer 5x, 2.5µl DTT 0.1M, 0.5µl unlabeled dNTPs 50x (dATP/dCTP/dGTP:dTTP 25:5), 1.5µl Cy5-dUTP 10mM or Cy3-dUTP 10mM (for Universal Human Reference RNA), 0.63µl RNasin Inhibitor and 1.25µl SuperScript II (200U/µl). The labeling reaction was incubated at room temperature for 10min and then incubated at 42ºC for 2h. Tubes were protected from direct light. 2.- Each fluorescent labeled cDNA was purified with CyScribe GFX Purification kit (Amersham) following manufacturer's instructions. 3.- The Cy3-labeled and Cy5-labeled cDNAs for each hybridization were combined into a single tube. 10µg of Human Cot-1 DNA (Invitrogen) were added. 4.- To precipitate each hybridization probe, 0.1 volumes of sodium acetate 0.1M and 2.5 volumes of absolute ethanol were added. 5.- Each probe was resuspended in 4µl of filtered EDTA 10mM ph 8 and denatured at 95ºC for 10min. 40µl of SlideHyb buffer 1x (Ambion), prewarmed at 65ºC, and blocking agents (mix of polyA and yeast tRNA, 10µg and 4µg in 2µl of final volume) were added.
Genomic DNA extracted from blood using conventional protocols for DNA extraction.
Label
Cy3
Label protocol
1.- DNA from each sample were primed with 3µl of random hexamers d(N)6 0.5mg/ml (Promega) at 70ºC for 10min. For each labeling reaction, a master mix was prepared: 5µl First Strand buffer 5x, 2.5µl DTT 0.1M, 0.5µl unlabeled dNTPs 50x (dATP/dCTP/dGTP:dTTP 25:5), 1.5µl Cy5-dUTP 10mM or Cy3-dUTP 10mM (for Universal Human Reference RNA), 0.63µl RNasin Inhibitor and 1.25µl SuperScript II (200U/µl). The labeling reaction was incubated at room temperature for 10min and then incubated at 42ºC for 2h. Tubes were protected from direct light. 2.- Each fluorescent labeled cDNA was purified with CyScribe GFX Purification kit (Amersham) following manufacturer's instructions. 3.- The Cy3-labeled and Cy5-labeled cDNAs for each hybridization were combined into a single tube. 10µg of Human Cot-1 DNA (Invitrogen) were added. 4.- To precipitate each hybridization probe, 0.1 volumes of sodium acetate 0.1M and 2.5 volumes of absolute ethanol were added. 5.- Each probe was resuspended in 4µl of filtered EDTA 10mM ph 8 and denatured at 95ºC for 10min. 40µl of SlideHyb buffer 1x (Ambion), prewarmed at 65ºC, and blocking agents (mix of polyA and yeast tRNA, 10µg and 4µg in 2µl of final volume) were added.
Hybridization protocol
3.- Hybridization: the probe (mix Cy3-labeled and Cy5-labeled cDNAs) was centrifuged at full speed for 5min at room temperature. Then it was applied (approx. 44µl, leave 2µl from the bottom of the tube behind in order to avoid transferring any sediment to the slide) to a clean cover-slip and place on the pre-hybridized microarray. The slide was placed in a hybridization chamber with the printed area and the cover-slip at the top, and was incubated overnight (15-20h) at 55ºC in an oven.
Scan protocol
Slides were scanned for Cy3 and Cy5 following the steps: 1.- Place each slide in a cassette as follows: orient the slide so that it is array-side down and to the left; take cassette flat side down, place on flat surface and push down on the two buttons; with the buttons pressed, insert the slide (keeping everything flat); release buttons when slide has been fully inserted. 2.- Place cassettes into carousel. The etched side should now face to the left, and you should be looking at the back of the slide. Note: the cassette must start with the first slot. The scanner recognizes an empty slot as a sign to stop scanning. Close the scanner top. 3.- Double-click on the Agilent Scanner icon. When it opens, the scanner will go through some initialization steps. Also, if the lasers are not fully warm, there will be a warning sign that will give an approximate time that the lasers will be ready. 4.- Enter your name under Operator and check to see that the controls are set for the following: output directory is D:\niehs; both Red and Green channels are checked and are set to 100%; the size is 10 microns. 5.- Click on Options: choose to scan default slide area under Scan Region Options; specify slide area to largest area under Default Scan Region; click on Instrument serial number under Automatic File Naming (it will then be highlighted). Choose and type in your identifier for that day. Leave the Slot Number as is. 6.- Fill out log in the Scanner Cassette Log with your name, date and the order you put your slides into the carousel. This is important to assure proper renaming after scanning is complete. Click on OK to close the Options window. 7.- Double check that all the settings are properly set and click on Scan. 8.- After scan is complete, you will need to change the names to format pxxsxx. To do this: with the right mouse button, click on the Start menu and choose Explorer. Navigate your way through the files by clicking on the D drive on the far left hand side of the screen. Double click on D drive then double click onniehs. This folder contains all of the scanned files. Locate your slides by looking for the identifier you used for the scan. Click once to highlight name and click once more to get a cursor in the file name. Type in the proper name using the log you filled out earlier. Once all of the names have been changed, highlight the newest slides and move them into the appropriate boxes on Witchfire. 9.- Once you are done, log out of the computer, take the cassettes out of the carousel and your slides out of the cassettes. Turn off scanner.
Description
The goal of this analysis is to see the copy number genes differential changed in lung cancer cell lines in relation with a control (normal females).
Data processing
Cy3 and Cy5 fluorescent signals were quantified using GenePix Pro 5.0 (Axon Instruments Inc.). Substandard spots were manually flagged and discarded for further analysis. The Cy5/Cy3 ratios were normalized to the median ratio value using the DNMAD tool, based on a print-tip loess method. The conditions for the normalization were: 1) use negative flags, that means that the flagged spots are not used for the normalization and are turned into missing values; 2) do not use background correction. The normalized and semi-logarithmic transformed data were preprocessing using GEPAS software. Conditions for data pre-processing were: 1) merge replicates (the Cy5/Cy3 ratios of replicated spots were averaged); 2) filter missing values in order to exclude for further analysis any gene with data missing in more than 10% of the samples.
