Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 85
can be achieved by performing the hybridization with 35% formamide (with the
same concentration of SSC and SDS) and at 42°C for at least 24 h.
11. Add SDS last to avoid precipitation.
12. Performing the first wash above room temperature (65°C) provides higher stringency
to increase specific to nonspecific hybridization signal. For oligonucleotide
arrays, perform the first wash at 42°C.
13. Work quickly to avoid air-drying (which will leave salt residue) between the last
wash and spin-dry steps.
14. High-quality linkers are crucial to several steps in the SAGE method. Linkers 1A,
1B, 2A, and 2B should be obtained gel-purified from the oligonucleotide synthesis
company. These oligonucleotides can be ordered from Integrated DNA Technologies
or other oligonucleotide synthesis companies.
15. The RNA volume should not exceed 100 µL.
16. A 1:10 dilution is often a good start.
17. Keep the supernatant; it contains your actual tags!
18. Use 1 µL of 1/50, 1/100, and 1/200 dilutions of ligation product per PCR reaction.
This step indicates the best dilution for the reactions. If the starting RNA is poor
quality, start out with a 1/25 dilution.
19. The appropriate cycle number is critical for isolating an adequate amount of ditag
DNA for SAGE. Too few cycles result in a low yield and may cause problems with
subsequent steps. Too many cycles give erratic results and also may result in low
yields. Therefore, trying various cycle numbers (e.g., 26, 28, or 30) to determine
the optimal number is recommended.
20. The DNA seeps out of the gel overnight. Do not leave for more than one night, or
the acrylamide can break down substantially. Do not put at –80°C.
21. Freeze/thaw cycles decrease the effectiveness of NlaIII. Freeze/thaw NlaIII only
once to insure optimal activity.
22. Do not run the voltage too high or your 26-bp product could melt.
23. This gel is thin and prone to rip. Be careful!
24. Developing of the gel should take approx 2 to 3 min and care should be taken not to
overexpose the gel (have the 1% acetic acid solution ready for quenching the reaction).
25. Stainer A and B are provided in the colloidal Coomassie kit provided by
Invitrogen.
Acknowledgments
A.M. is supported by an American Association for Cancer Research-PanCAN
Career Development Award, the Lustgarten Foundation for Pancreatic Cancer
Research, and National Cancer Institute R01CA113669.
References
1. Yeo, T. P., Hruban. R. H., Leach, S. D., et al. (2002) Pancreatic cancer. Curr.
Probl. Cancer 26, 176–275.
2. Takaori, K., Hruban, R. H., Maitra, A., and Tanigawa, N. (2004) Pancreatic
intraepithelial neoplasia. Pancreas 28, 257–262.