1. miRNA Platform Overview The Agilent miRNA Microarray System A New Microarray-based Tool for Profiling Human miRNAs

2. miRNA Platform Overview Choose from a comprehensive set of microarray applications DNA RNA Splice Variants ChIP Elucidate the role that protein-DNA interactions play in transcription, replication, modification and repair Perform global interrogations of the transcriptome and identify alternative splice forms GX Explore gene transcription on a genome-wide basis mRNA From one-dimensional with multiple applications … aCGH Conduct high- resolution, genome-wide profiling of DNA copy number changes Copy number CH 3  Discover and monitor epigenetic modifications MethylationTranscription Factors mRNA isoforms miRNA Study microRNAs and the role they play in gene regulation miRNA Profiling to multi-dimensional …

3. miRNA Platform Overview Analysis within one context Splice Variants ChIP Gene Expression aCGH The Integrated Agilent Platform CH 3 miRNA

4. miRNA Platform Overview miRNA Scientific Background and Importance  Definition: 19-30 nucleotide long single- stranded RNAs that post-transcriptionally regulate gene expression  Key regulators of gene expression, development, proliferation, differentiation, and apoptosis  May regulate >30% of human genes  Current Sanger miRBASE Release 9.1 has 4361 entries, 474 identified in humans  Projected $100M to be awarded by the NIH for miRNA-related research in 2008*  Discovery of new miRNAs is ongoing … *Source: NIH CRISP database at http://crisp.cit.nih.gov/ Cancer Genomics: Small RNAs with big impacts from Nature 435: 745-746 (9 June 2005)

5. miRNA Platform Overview miRNAs in Cancer  Cancer cells generally have lower levels of miRNAs than normal cells  Depending on the genes they regulate, miRNAs can function as either an oncogene or tumor suppressor  miRNA profiles can be used to classify human tumors (Lu et al., Nature 435, 834 (2005) - Profiling of 217 miRNAs from 334 tumors yielded highly accurate classification - Classifications reflects developmental lineages and tissue of origin  Alterations of miRNA expression have been observed in a number of cancers - Colorectal, pituitary, lung, breast, B-cell lymphoma, glioblastoma, others - In several cancers, specific miRNA signatures are associated with prognosis  Using CGH, shown that miRNA loci have a high frequency of genomic alterations in human cancers (Zhang et al, PNAS 103, 9136 (2006)

6. miRNA Platform Overview Growth of miRNA Publications Source: PubMed search with terms “miRNA” or “microRNA” Publication Number

7. miRNA Platform Overview The Agilent miRNA Profiling System/Workflow total RNA (100 ng) Direct Label with Cyanine 3-pCp Hybridize on Microarray miRNA profile labeled RNA Data analysis with GeneSpring GX 8-plex microarray

8. miRNA Platform Overview Agilent miRNA Platform Highlights  Low sample input - 100 ng of total RNA  No small RNA isolation required  Simple one-tube protocol – results in < 2 days  High sequence and size specificity – mature miRNAs  High sensitivity – Detection limit <0.1 αmol  Broad linear dynamic range (>4 logs)  Replicate probes per array (20 per miRNA)  One-color analysis Enabled by Agilent miRNA Probe Design and Direct Labeling Methodology

9. miRNA Platform Overview Direct and Sensitive miRNA Profiling Back to Applications Platform Hui Wang, PhD Senior Scientist Agilent Technologies

10. miRNA Platform Overview Probe Design 1. Probe connected to array surface by stilt 2. Add G residue to hybridize to target label C provides stability. 3. Adjust probe size to match Tm. 4. Hairpin structure provides specificity by destabilizing hybridization to larger, non-target RNAs. Empirical selection of final probes by microarray validation

11. miRNA Platform Overview Specificity to Distinguish Homologous miRNAs hsa-let-7 family

12. miRNA Platform Overview Purified Small RNA (Cy3) Total RNA (Cy3) Direct miRNA Measurement From Total RNA Data shown are background-subtracted signals with no filtering or normalization. Each miRNA has signals from multiple probes.

13. miRNA Platform Overview miRNA Profiles Using 100ng Total RNA Placenta Brain ReproducibilityDifferential Expression

14. miRNA Platform Overview Quantitative RT-PCR (qPCR) reactions (orange bars) and microarray hybridizations (blue bars) were run in quadruplicate for each miRNA species. Microarray Data qPCR Data Correlation between microarray and quantitative RT-PCR results

15. miRNA Platform Overview We have shown for the first time that let-7 expression is frequently reduced in lung cancers and that alterations in the miRNA expression may have a prognostic impact on the survival of surgically treated lung cancer patients. Agilent miRNA arrays give us the comprehensive miRNA expression profile with excellent performance on sensitivity and accuracy. I expect that the studies of Agilent miRNA array may ultimately provide a foundation for a new paradigm of the involvement of miRNA in human oncogenesis. Dr. Takashi Takahashi Professor of Oncology Molecular Carcinogenesis Nagoya University Response from Early Access Customer

16. miRNA Platform Overview miRNA Microarray System Components Built on the core microarray platform from Agilent  Human miRNA Microarray kit (3 slides, 24 samples total) -Contains probes for 474 human miRNAs in the Sanger miRBASE 9.1 -8-plex slide (8 independent microarrays per glass slide)  Cyanine 3-pCp labeling reagent [24 reactions]  Complete protocols for labeling, hybridization, scanning, and data analysis Release 1.0 products available May 2007:

17. miRNA Platform Overview Agilent’s miRNA Product Roadmap  Release 1.0 – May 2007  Human 1.0 microarray  pCp-Cy3 labeling reagent  Feature Extraction 9.5  Complete protocol and source information for other reagents  Release 2.0  Human 2.0 microarray  Mouse and Rat 1.0 microarrays  Labeling kit with all required reagents  Future product releases  Update microarray content with discovery of new miRNAs  Spike-in controls  Enabling custom miRNA content  Enhance miRNA-related features in GeneSpring GX Your Genomics Story Confident miRNA Profiling Agilent’s DNA Microarray Platform

18. miRNA Platform Overview Questions please.. Thank You!!