Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 114

5. In GCOS, press the “start” icon, and while the scanner is performing its automated
tasks, click on “view” and “scan in progress” to visualize the results once scanning
begins.
6. A successful scan will create the *.DAT and *.CEL files. The *.CEL files are
now ready for analysis to create the *.CHP file for subsequent data analysis.

4. Notes
1. Because microarray analysis requires the use of fluorescence, the lysis buffer
must be chosen carefully. For instance, many lysis reagents contain phenol and
chloroform. Residual phenol and or chloroform can interfere with fluorescent
labeling. This usually equates to less signal and more background and hence
difficulty in interpreting microarray results. The lysis buffer RLT (provided with
the RNeasy Mini Kit) does not interfere with subsequent labeling of the samples.
2. It is difficult to determine RNA quantity from S. aureus during S. aureus -PMN
interactions because the S. aureus RNA isolation procedure will also isolate PMN
RNA. Because of the development of very specific oligonucleotide-based bacterial
gene chips, it is no longer necessary to remove host RNA (3) . RNA isolated as
described above typically yields 100-200 μg/mL of total RNA (RNA from S.
aureus and human PMNs). RNA can be measured by absorbance at 260 nm on
a spectrophotometer (1 absorbanceU=40μg/mL RNA). The ratio of 260/280
should be approximately 2.0. Alternatively, a fluorescence-based assay such as
Quant-iT RiboGreen RNA assay (Invitrogen Life Technologies) can be used. This
assay requires the use of a microplate spectrofluorometer (e.g., Molecular Devices
Gemini XPS, Sunnyvale, CA). Quantifying cDNA only provides an estimate of
how much of the sample is S. aureus cDNA. Like RNA, cDNA can be measured
by absorbance at 260 nm (1 absorbanceU=33μg/mL of single-stranded DNA).
Alternatively, cDNA can be quantitated using a fluorescence-based assay such as
Quant-iT OliGreen ssDNA assay (Invitrogen Life Technologies).
3. Lot-to-lot variation of DNase I enzyme activity is possible. A simple titration
assay can be performed to independently determine DNase I activity. Fragmented
samples (200 ng) can be loaded onto a 4-20% acrylamide gel and stained with
SYBR Gold. Size of fragmented cDNA will be 50-200 base pairs.

4. Conditions for the terminal labeling reaction are determined by the format of
the specific Affymetrix GeneChip being used. The reaction described above is
optimized for the Standard (49) Format.
5. In order to fill the 49 format GeneChip without air bubbles, approximately 300 μL
of wash A is required. Place the chip flat on the bench with the septa facing up.
Place a 200 μL pipette tip in a septum and fill a 200 μL pipette with wash A. Hold
the GeneChip parallel to the floor and insert the full pipette into the open septum.
Dispense only about 180 μL of the 200 μL and remove the pipette assuring no
air bubble will be in the septa region. Switch to a new tip, then load another 200
μL of wash A. Making sure no air bubbles are at the tip of the pipette, insert it
once again into the open septum and hold the chip perpendicular to the floor with

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