Isolation and Culture of Vascular Smooth Muscle Cells

3.3 Microarray Scanning

cartridge with 250 μL of the non-stringent wash buffer. The array is now ready for washing and staining in the fluidics station. As the station can only process four arrays at a time, other arrays to be processed can be stored temporarily at 4 °C for up to 3 h. 5. The fluidics station needs to be primed to ensure the lines are filled with the appropriate buffers and is ready for running protocols. The non-stringent wash buffer should be filled in the

Wash A buffer reservoir on the machine. The stringent wash buffer should be filled in the Wash B buffer reservoir. Run the

Prime_450 maintenance protocol with empty microcentrifuge tubes in the stain holder positions 1, 2, and 3. 6. After priming, the fluidics station is ready to accept arrays for washing and staining. Using the proper protocol enter the sample file name, the array name, and the probe array type.

Select the fluidics protocol script for processing the arrays.

For our experiments, the fluidics script used was the EukGE-

WS2v5_450. Follow instructions on the LCD window on the fluidics station for loading the array into the machine and for loading of the sample holders. There are three sample holders on the machine module. Place one vial containing 600.0 μL

SAPE stain solution in sample holder 1. Place one vial containing 600.0 μL antibody solution in sample holder 2. Place one vial containing 600.0 μL SAPE stain solution in sample holder 3. Press down on the needle lever to snap needles into position which will start the run. 7. When the protocol is complete, the LCD window will display the message EJECT & INSPECT CARTRIDGE. Press down on the cartridge lever to the eject position and remove the array. Do not engage the washblock until the array has been inspected for the presence of bubbles or air pockets. If the array has no bubbles, it is ready for scanning. If there are bubbles present, reinsert the array back into the washblock probe array holder and engage the washblock. The array will be drained and refilled. Recheck the array for any bubbles and when none are present, continue on scanning the array. Engage all washblocks for the fluidics station to continue to complete the protocol and prime for the next wash protocol. 8. If the arrays are not scanned immediately following washing and staining, they can be stored at 4 °C, in the dark, until ready for scanning for a maximum of 24 h. 9. A shutdown protocol should be run on the fluidics station at the end of the daily session.

The scanning of the Affymetrix GeneChip® Rat Genome 230 2.0 Arrays are automated using the Affymetrix GeneChip® Scanner 3000. The sample file created using the GCOS software (or the

3.4 Scanned Image Analysis

updated AGCC software) will be referenced for scanning of the array. After washing and staining of the array, the sample file will be available in the scanner software. Samples were scanned according to the Affymetrix 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).

The manual provides step-by-step directions for using the AGGC software to operate the Affymetrix GeneChip® Scanner 3000. Once samples are washed and stained, they can be scanned using the manufacturer’s protocol (see Note 9).

The output of the scanning creates two files for each array. The first file created during scanning is the raw pixelated image of the array and is referred to as a .DAT file. The Affymetrix software saves this file and aligns a grid onto the image to locate and identify the probe cell features. The control oligonucleotide B2 included in the hybridization cocktail allows for this alignment. The software takes each pixelated probe feature from the .DAT file and automatically calculates a single intensity value for each probe feature (non-pixelated) to create a second image file. This file contains all of the probe cell intensity data that is referred to as the .CEL file. It is the most important file that is generated and is used by Affymetrix or other third-party software to determine single gene intensity values based on their respective probe intensities.

The Affymetrix expression software, either GCOS or the Affymetrix Expression Console, can be used to visualize the .CEL files for verification of a problem-free array and can be used to analyze the data to generate reports to ensure the microarrays in an experiment are suitable for subsequent data analysis.

Affymetrix created their gene expression arrays utilizing sets of 25-mer oligonucleotide probes designed specifically for each individual gene. An individual gene probe set is composed of 11 perfect match primers (PM) and 11 mismatch primers (MM) (the middle base of the 25-mer perfect match is changed). In other words, each gene has a set of 22 individual probes whose intensities are available to be used for analysis in determining a single intensity of the specified gene using Affymetrix software or other third-party software.

A visual inspection of the array should be done to determine all areas of the array suitable for analysis. 1. The array image should be clear of “bubbles” (areas where there was no hybridization due to air bubbles trapped in the array during hybridization), scratches, or other problematic areas. If these areas are present, they need to be masked and not used in subsequent analysis.

2. The image should have an overall “equal” image intensity without large areas of extreme intensity. 3. When the grid is applied on the array, the border of the array should be examined for a checkerboard appearance in each corner of the array and an alternating pattern on the border edges of the entire array that are aligned within the grid boxes.

This alignment uses the hybridization of the control oligonucleotide B2 at these specific probe cells on the array. 4. In the center section of the array image, a small cross should be present which is also a result of the control oligonucleotide B2 hybridization and should be aligned within the grid boxes. 5. If alignment does not automatically occur directly, manual adjustment can be made to the grid using an Affymetrix manual protocol. In our experience over 10 years, we have never had to adjust any grid alignments, nor have we had to use any masking of problematic areas of the microarrays.

Prior to data analysis, the arrays to be included in an experiment must be evaluated to ensure they are within certain parameters to be considered comparable and suitable for data analysis. To do this, the user will rely on a report file (.RPT) that can be generated with GCOS or the Affymetrix Expression Console software (see Note 10). In either GCOS or the Expression Console, the researcher will use the MAS 5.0 algorithm to analyze each array independently in order to obtain a report regarding the performance of the array. It is important to identify any obvious problems at this point before submitting data from the experimental set of arrays to a multichip analysis method, either in the Expression Console or with third-party software. For the generation of a MAS 5.0 algorithm .RPT file, the researcher should use the GCOS software to create a tabular formatted analysis results file or .CHP file. The software allows for scaling and normalization of each array as it is analyzed by the MAS 5.0 algorithm to create the .CHP file. Our experiments used a scaling setting for all probe sets (set at 150) to achieve a scale factor for each array which will be referred to below. No normalization adjustments were made to the data and Affymetrix default settings were used for all other limits. Once a .CHP file is created, the software can then be used to create a report (.RPT) file.

The report file contains information to evaluate performance of the array. Various laboratories may use different limit settings for deciding which arrays can be compared with confidence. The top heading of each report file lists the file name, probe array type, and the algorithm used. The following list contains those parameters, which in our hands provide confident assurance to continue on with the arrays for multichip analysis.