Bacterial artificial chromosome-emulation oligonucleotide arrays for targeted clinical array-comparative genomic hybridization analyses.

PURPOSE: The goal of this work was to test the ability of oligonucleotide-based arrays to reproduce the results of focused bacterial artificial chromosome (BAC)-based arrays used clinically in comparative genomic hybridization experiments to detect constitutional copy number changes in genomic DNA. METHODS: Custom oligonucleotide (oligo) arrays were designed using the Agilent Technologies platform to give high-resolution coverage of regions within the genome sequence coordinates of BAC/P1 artificial chromosome (PAC) clones that had already been validated for use in previous versions of clone arrays used in clinical practice. Standard array-comparative genomic hybridization experiments, including a simultaneous blind analysis of a set of clinical samples, were conducted on both array platforms to identify copy number differences between patient samples and normal reference controls. RESULTS: Initial experiments successfully demonstrated the capacity of oligo arrays to emulate BAC data without the need for dye-reversal comparisons. Empirical data and computational analyses of oligo response and distribution from a pilot array were used to design an optimized array of 44,000 oligos (44K). This custom 44K oligo array consists of probes localized to the genomic positions of >1400 fluorescence in situ hybridization-verified BAC/PAC clones covering more than 140 regions implicated in genetic diseases, as well as all clinically relevant subtelomeric and pericentromeric regions. CONCLUSIONS: Our data demonstrate that oligo-based arrays offer a valid alternative for focused BAC arrays. Furthermore, they have significant advantages, including better design flexibility, avoidance of repetitive sequences, manufacturing processes amenable to good manufacturing practice standards in the future, increased robustness because of an enhanced dynamic range (signal to background), and increased resolution that allows for detection of smaller regions of change.

Heterogeneity of mammary lesions represent molecular differences.

BACKGROUND: Human breast cancer is a heterogeneous disease, histopathologically, molecularly and phenotypically. The molecular basis of this heterogeneity is not well understood. We have used a mouse model of DCIS that consists of unique lines of mammary intraepithelial neoplasia (MIN) outgrowths, the premalignant lesion in the mouse that progress to invasive carcinoma, to understand the molecular changes that are characteristic to certain phenotypes. Each MIN-O line has distinguishable morphologies, metastatic potentials and estrogen dependencies. METHODS: We utilized oligonucleotide expression arrays and high resolution array comparative genomic hybridization (aCGH) to investigate whole genome expression patterns and whole genome aberrations in both the MIN-O and tumor from four different MIN-O lines that each have different phenotypes. From the whole genome analysis at 35 kb resolution, we found that chromosome 1, 2, 10, and 11 were frequently associated with whole chromosome gains in the MIN-Os. In particular, two MIN-O lines had the majority of the chromosome gains. Although we did not find any whole chromosome loss, we identified 3 recurring chromosome losses (2F1-2, 3E4, 17E2) and two chromosome copy number gains on chromosome 11. These interstitial deletions and duplications were verified with a custom made array designed to interrogate the specific regions at approximately 550 bp resolution. RESULTS: We demonstrated that expression and genomic changes are present in the early premalignant lesions and that these molecular profiles can be correlated to phenotype (metastasis and estrogen responsiveness). We also identified expression changes associated with genomic instability. Progression to invasive carcinoma was associated with few additional changes in gene expression and genomic organization. Therefore, in the MIN-O mice, early premalignant lesions have the major molecular and genetic changes required and these changes have important phenotypic significance. In contrast, the changes that occur in the transition to invasive carcinoma are subtle, with few consistent changes and no association with phenotype. CONCLUSION: We propose that the early lesions carry the important genetic changes that reflect the major phenotypic information, while additional genetic changes that accumulate in the invasive carcinoma are less associated with the overall phenotype.

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray.

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance.

In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling.

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.