Measurement of Superoxide Production and NADPH Oxidase

Fig. 1 Representative absorption spectrum of a good-quality RNA sample

2.13 Microarray Hybridization, Washing and Staining of Sample Targets

Several cDNA preparation kits are available from commercial vendors such as Bio-Rad, Life Technologies, and QIAGEN. 3. Use the RNA for preparation for Affymetrix arrays: In this case, Affymetrix has a unique procedure for one-cycle cDNA synthesis, target labeling, and cRNA preparations. Follow the

Affymetrix manual, which is quite detailed and self-explanatory for the most part.

Please note that besides Affymetrix, there are a number of additional commercial microarray service providers for your hybridization experiments (Table 1).

For experiments described in our work [9], we used the Affymetrix GeneChip® microarrays (http://www.affymetrix.com/estore/). The Affymetrix system used consisted of the Affymetrix GeneChip® Hybridization Oven 640, the Affymetrix GeneChip® Fluidics Station 450, and the Affymetrix GeneChip® Scanner 3000 6G. The software utilized was the Affymetrix GeneChip® Operating Software (GCOS), version 1.4. Manuals used for protocols were the GeneChip® Expression Analysis Technical Manual (catalog number 702232, Revision 3) (http://media.affymetrix.com/ support/downloads/manuals/expression_analysis_manual.pdf) and the GeneChip® Expression, Wash, Stain and Scan User Manual (catalog number 702731, Revision 3) (http://media.affymetrix. com/support/downloads/manuals/wash_stain_scan_cartridge_ arrays_manual.pdf). For user-prepared protocols, in the Analysis

Affymetrix ® platform Illumina ® platform Illumina ® platform RT 2 ProfilerTM PCR Array platform Agilent gene expression microarray platform NimbleGen platform Illumina ® platform

Table 1 Details of some of the microarray service providers in the United States Website

http: // www.affymetrix.com/estore/ http: // www.illumina.com/ http: // www.star-array.com/technology.html http: // www.sabiosciences.com/RTPCR.php http: // www.genomics.agilent.com/CollectionOverview.aspx?P ageType=Application&SubPageType=ApplicationOverview &PageID=103 http: // www.nimblegen.com/products/expression/index.html http: // www.arraystar.com/index.asp http: // www.otogenetics.com/rnaseq_page_010812.htm

Microarray service provider

Affymetrix 3420 Central Expressway Santa Clara, CA 95051 USA Illumina, Inc. 5200 Illumina Way San Diego, CA 92122 USA Star Array Unit 223, Innovation Center BLK 2, 18 Nanyang Drive, Singapore 637723 QIAGEN Inc. 27220 Turnberry Lane Valencia, CA 91355 USA Agilent 5301 Stevens Creek Blvd Santa Clara, CA 95051 USA Roche NimbleGen, Inc. 500 South Rosa Road Madison, WI 53719 USA Arraystar Otogenetics

Technical manual, refer to Appendix B, and in the Wash, Stain and Scan manual, refer to Chapter 2.

In recent years, Affymetrix has updated both the sample preparation protocols and the system equipment. For example, Affymetrix has since discontinued the GCOS software and now uses the Affymetrix GeneChip® Command Console (AGCC) (http://www.affymetrix.com/esearch/search.jsp?pd=131429& N=4294967292) with the GeneChip® Expression Console (http: //www.affymetrix.com/esearch/search.jsp?pd=131414&N= 4294967292) to generate similar data. All experimental procedures performed in this manuscript utilized the GCOS software (see Note 1). The Affymetrix scanner has been upgraded to a 3000 7G model which scans with an increased resolution of 7 μm and allows for different types of Affymetrix microarrays to be utilized in research. It is highly recommended that the researcher refer to Affymetrix for the most updated expression analysis methodology (http://www.affymetrix.com/estore/).

Important aspects for designing a microarray study: Most microarray experiments are run with a minimum of six samples (three control and three experimental) to achieve data that can be mined with statistical programs. The longest step of the process is the overnight (16 h) hybridization. For washing and staining following hybridization, the fluidics station can process four arrays at a time, and normally, eight arrays can be run in 1 day with subsequent scanning of the arrays. If the user is running more than eight samples, it is recommended that all samples be processed at one time to create all of the sample hybridization cocktails. Once created, they can be stored at −20 °C until ready for hybridization to the arrays.

For example, assume an experiment consists of 24 arrays for processing: 12 control samples (sample #’s 1–12) and 12 experimental samples (sample #’s 13–24). As eight samples can be run per day, the overall work should be planned as outlined in the following timeline. It is highly recommended that the user does not process only samples from the same group on the same day. In our example, on day 1, control sample #’s 1–4 and experimental sample #’s 13–16 are processed.

Experimental timeline example for 24 samples

Day 1 Day 2 Day 3 Day 4

Thaw #1 - #4 #13 - #16

Hybridize #1 - #4 #13 - #16 Process Wash Stain Scan #1 - #4 #13 - #16

Thaw #5 - #8 #17 - #20

Hybridize #5 - #8 #17 - #20 Process Wash Stain Scan #5 - #8 #17 - #20

Thaw #9 - #12 #21 - #24

Hybridize #9 - #12 #21 - #24 Process Wash Stain Scan #9 - #12 #21 - #24