Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 69
3.1.5. Washing Microarrays After Hybridization
After hybridization, wash the DNA microarrays to remove unbound labeled
sample. The following three sequential wash steps should be performed using a
slide rack, transferring among three separate glass staining dishes, each containing
volumes of 350–400 mL, with gentle agitation:
• Wash 1: 2X SSC/0.03% SDS (see Note 11), at 65°C (see Note 12), 5 min.
• Wash 2: 1X SSC, room temperature, 5 min.
• Wash 3: 0.2X SSC, room temperature, 5 min.
After the third wash, DNA microarrays should be spun dry by using a
desktop centrifuge, at 500g at room temperature for 5 min (see Note 13).
3.1.6. Microarray Imaging, Data Reduction, and Analysis
After hybridization, DNA microarrays should be scanned in dual wavelengths
by using a GenePix 4000B (Molecular Devices) scanner, or equivalent.
GenePix software, or equivalent, should be used to extract fluorescence intensity
ratios for each DNA element on the DNA microarray. To compensate for
variable labeling efficiencies, fluorescence ratios can be normalized such that
the average fluorescence ratio for all DNA elements on the array is set to 1 (i.e.,
“global” normalization). Microsoft Excel, or any of several more sophisticated
commercial or academic microarray databases (43), can be used to manipulate
and analyze microarray data.
3.2. Serial Analysis of Gene Expression
3.2.1. Kinasing Reaction for Linkers (See Note 14)
The linkers need to be “kinased” as described in the NEB T4 polynucleotide
kinase instructions (New England Biolabs). Briefly,
1. Dilute Linker 1B, 2B, 1A, and 2A to 350 ng/µL.
2. Incubate kinase reaction solution per NEB instructions at 37°C for 30 min and
heat inactivate at 65°C for 10 min.
3. Mix 9 µL of Linker 1A (350 ng/mL) into the 20 µL of kinased Linker 1B prepared
above (final conc. 200 ng/µL) to generate “1AB.”
4. Then, mix 9 µL of Linker 2A (350 ng/µL) into the 20 µL of kinased Linker 2B
prepared in Step 2 (final conc. 200 ng/µL) to generate “2AB.”
5. Anneal both tubes of linkers with the following incubations: 1) 95°C for 2 min,
2) 65°C for 10 min, 3) 37°C for 10 min, 4) room temperature for 20 min, 5) Store
at –20°C. Store at –20°C.
6. Test the anneal mixes. Kinasing should be tested by self-ligating both mixes and
incubating at 37°C for 30 min.
7. Analyze on a 1.5-mm 12% PAGE-TAE gel. The ligated dimers should run at 80 bp,
and the monomers should be run at 40 bp. Load all of the self-ligated linkers, and