Target Discovery and Validation Reviews and Protocols

62 Beaty et al.
oligonucleotide microarray approach (39). This tight junction molecule also has
been used for targeting immunotoxins to pancreatic cancer cells (40,41) as well
for imaging purposes (35). In transcriptomic studies, proteomic analysis of pancreatic
juice samples has led to the identification of regenerating islet-derived
3 alpha (REG3A), also known as pancreatitis-associated protein, as overexpressed
in cancer samples compared with nonneoplastic pancreatic juice (21).
The selective overexpression of REG3A in neoplastic juice samples implies that
this protein can be used as an early detection biomarker for pancreatic cancer.
2. Materials
2.1. DNA Microarrays
1. 20X Standard saline citrate (SSC) solution.
2. 10% Sodium dodecyl sulfate (SDS) solution.
3. Bovine Serum Albumin (BSA) (cat. no. A7888, Sigma); prepare a 10 mg/mL BSA
stock solution, filtered.
4. Ethanol, 95% (Gold Shield Chemical Co.).
5. Diamond scribe (cat. no. 52865-005, VWR).
6. Slide humidifying chamber (cat. no. H6644, Sigma).
7. Heating block (cat. no. 13259-050, VWR), invert so solid metal surface faces up.
8. Ultraviolet source (Stratalinker 2400, Stratagene; or equivalent).
9. Pyrex dish for denaturing spotted cDNA on arrays (cat. no. 08-741F, Fisher).
10. Slide rack and glass staining dish (Wheaton).
11. Desktop centrifuge (Beckman Allegra 6R, or equivalent), with microtiter plate
carriers.
2.1.1. Labeling mRNA
1. RNase-free H 2 O (cat. no. 9935, Ambion; or equivalent).
2. Anchored oligo(dT) reverse transcriptase (RT) primer (T 20 VN, custom primer from
Operon; or equivalent; HPLC purify); prepare 2.5 µg/µL stock in TE 8.0 buffer.
3. Superscript II reverse transcriptase (cat. no. 18064-014, Invitrogen, Carlsbad, CA).
4. 5X First-Strand buffer (cat. no. Y00146, Invitrogen).
5. 0.1 M Dithiothreitol DTT (Y00147, Invitrogen).
6. RNasin (cat. no. N2511, Promega).
7. 50X dNTP mix: 10 mM dTTP, 25 mM each dGTP, dATP, dCTP, in TE 8.0 buffer);
prepare from ultrapure dNTP solutions; GE Healthcare).
8. Cy5-dUTP and Cy3-dUTP (cat. nos. PA55022 and PA53022, GE Healthcare).
9. 0.5 M EDTA.
10. Water bath(s) (70, 42, and 37°C).
2.1.2. Hybridization of Labeled Sample
1. RNase-free H 2 O (cat. no. 9935, Ambion; or equivalent).
2. PolyA RNA (cat. no. P9403, Sigma). Prepare 10 µg/µL stock solution in TE 8.0 .