Target Discovery and Validation Reviews and Protocols

84 Beaty et al.
search in. A widely used nonredundant database is RefSeq (53) (http://
www.ncbi.nlm.nih.gov/RefSeq/) from National Center for Biotechnology
Information, which contains 1,310.800 entries from 2780 organisms. One very
important aspect of the final analysis is verification of the data obtained from
the database searches. Far from all proteins retrieved from the database search
are correct. Peptides identified by the Mascot search engine with a peptide
score under 30–40 are usually false positives and have to be discarded from the
data set. In cases where protein identification is based on a single peptide, special
caution has to be taken. The amino acid sequence identified by the Mascot
search engine has to be verified manually to make sure that the MS/MS spectrum
truly corresponds to the peptide sequence predicted by Mascot. Manual
validation is therefore essential to avoid a large number of false positives in the
list of identified proteins.
4. Notes
1. After processing, the array spots will no longer be visible, so demarking the
boundaries is important for later positioning the labeled sample and cover slip
correctly.
2. Rehydration should produce spot sizes that are enlarged by approx 20%, with the
DNA distributed more uniformly within spots.
3. Bring ddH2O to a boil in the Pyrex dish and then remove from heat. Add microarrays
in slide rack immediately after the boiling (bubbling) has subsided.
4. For processing oligonucleotide arrays, UV-crosslink by using 70 mJ, wash arrays
in 0.2% SDS at room temperature for 10 min, wash three times in ddH2O at room
temperature for 5 min each, rinse in 95% EtOH for 1 min, and then spin-dry in
centrifuge at 500 rpm.
5. Often by convention, the test sample is labeled with Cy5, whereas the reference
sample is labeled with Cy3.
6. Centrifuge times are estimates. If necessary, here and in subsequent Microcon
steps, spin in additional 1-min increments until volume of retained solution is
approx 20 µL.
7. This additional wash step serves to further remove unincorporated fluorescent
nucleotides. If labeling is successful, the retained labeled cDNA mixture should
be light blue.
8. PolyA and human Cot-1 preannealing serves to block undesirable hybridization to
polyA tails and highly repetitive DNA, respectively, contained in a subset of cDNA
microarray elements. Yeast tRNA functions to block nonspecific hybridization.
9. A total volume of 40 µL for the hybridization solution is appropriate when using
a 22- × 60-mm cover slip. If using a different-sized cDNA microarray and cover
slip, adjust the total volume of hybridization solution accordingly, while maintaining
final SSC and SDS concentrations.
10. The described hybridization protocol is optimized for cDNA microarrays. For
oligonucleotide microarrays, improved hybridization sensitivity and specificity