Karen W. Shannon

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

[2] The Agilent In Situ‐Synthesized Microarray Platform

Abstract Microarray technology has become a standard tool in many laboratories. Agilent Technologies manufactures a variety of catalog and custom long‐oligonucleotide (60‐mer) microarrays that can be used in multiple two‐color microarray applications. Optimized methods and techniques have been developed for two such applications: gene expression profiling and comparative genomic hybridization. Methods for a third technique, location analysis, are evolving rapidly. This chapter outlines current best methods for using Agilent microarrays, provides detailed instructions for the most recently developed techniques, and discusses solutions to common problems encountered with two‐color microarrays.

Estimation of the confidence limits of oligonucleotide array-based measurements of differential expression

Microarrays can be used to simultaneously measure the differential expression states of many mRNAs in two samples. Such measurements are limited by systematic and random errors. Systematic errors include labeling bias, imperfect feature morphologies, mismatched sample concentrations, and cross-hybridization. Random errors arise from chemical and scanning noise, particularly for low signals. We have used a combination of fluor-exchanged two- color labeling and improved normalization methods to minimize systematic errors from labeling bias, imperfect features, and mismatched sample concentrations. On-array specificity control proves and experimentally proven probe design algorithms were used to correct for cross- hybridization. Random errors were reduced via automated non-uniform feature flagging and an advanced scanner design. We have scored feature significance, using established statistical tests. We have then estimated the intrinsic random measurement error as a function of average probe signal via sample self-comparison experiments (human K-562 cell mRNA). Finally, we have combined all of these tools in the analysis of differential expression measurements between K-562 cells and HeLa cells. The results establish the importance of the elimination of systematic errors and the objective assessment of the effects of random errors in producing reliable estimates of differential expression.