Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 63
3. Yeast tRNA (cat. no. 15401-011, Invitrogen). Prepare 5 µg/µL stock solution in
TE 8.0 .
4. Human Cot-1 DNA (cat. no. 15279-011, Invitrogen). Supplied as 1 µg/µL stock
solution.
5. 20X SSC.
6. 10% SDS.
7. Microcon-30 filters (Amicon).
8. Hybridization chambers (Corning; Monterey Industries, http://www.montereyindustries.com;
or equivalent).
9. Glass microscope slide cover slips, 22 × 60 mm (Fisher).
10. Slide rack and three glass staining dishes (Wheaton).
11. Desktop centrifuge (Beckman Allegra 6R, or equivalent), with microtiter plate
carriers.
12. Water bath (65°C).
2.1.3. Data Reduction and Analysis
1. GenePix 4000B scanner (Molecular Devices), or equivalent, for imaging microarrays
after hybridization.
2. GenePix software (Molecular Devices), or equivalent, for data extraction (i.e.,
matching pixels to DNA spots; calculating fluorescence ratios).
3. Microsoft Excel, or more specialized microarray databases (e.g., AMAD,
http://www.microarrays.org/software.html), for data storage, retrieval, and analysis.
2.2. Serial Analysis of Gene Expression
The materials required for SAGE can be divided into 13 discrete steps that
are listed stepwise. Note that some reagents are required in multiple steps.
2.2.1. Kinasing Reaction for Linkers (Step 1)
1. Linker sequences.
a. Linker 1A (obtain gel-purified).
5′-TTT GGA TTT GCT GGT GCA GTA CAA CTA GGC TTA ATA GGG ACA
TG-3′.
b. Linker 1B (obtain gel-purified).
5′-TCC CTA TTA AGC CTA GTT GTA CTG CAC CAG CAA ATC C[amino
mod. C7]-3′.
c. Linker 2A (obtain gel-purified).
5′-TTT CTG CTC GAA TTC AAG CTT CTA ACG ATG TAC GGG GAC
ATG-3′.
d. Linker 2B (obtain gel-purified).
5′-TCC CCG TAC ATC GTT AGA AGC TTG AAT TCG AGC AG[amino mod.
C7]-3′.
2. NEB T4 polynucleotide kinase (10 U/µL; cat. no. 201S, New England Biolabs).