miRNA Platform Overview

The Agilent miRNA Microarray System

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A New Microarray-based Tool for

Profiling Human miRNAs

miRNA Platform Overview

Choose from a comprehensive set of microarray applications

DNA RNA

Splice VariantsChIP

• 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

CH3

Discover and monitor epigenetic modifications

Methylation Transcription Factors

mRNA isoforms

miRNA

• Study microRNAs and the role they play in gene regulation

miRNA Profiling

to multi-dimensional …

miRNA Platform Overview

AnalysisAnalysiswithinwithin

one contextone context

Splice VariantsChIP

GeneExpressionaCGH

The Integrated Agilent Platform

CH3 miRNA

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)

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 oncogeneor 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 alterationsin human cancers (Zhang et al, PNAS 103, 9136 (2006)

miRNA Platform Overview

Growth of miRNA Publications

Source: PubMed search with terms “miRNA” or “microRNA”

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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 withGeneSpring GX

8-plex microarray

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

miRNA Platform Overview

Direct and Sensitive miRNA Profiling

Back to Applications

Platform

Hui Wang, PhD Senior Scientist

Agilent Technologies

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

miRNA Platform Overview

Specificity to Distinguish Homologous miRNAsmiRNAs

hsa-let-7ahsa-let-7bhsa-let-7chsa-let-7dhsa-let-7ehsa-let-7fhsa-let-7ghsa-let-7i

UGAGGUAGUAGGUUGUAUAGU UUGAGGUAGUAGGUUGUGUGGUUUGAGGUAGUAGGUUGUAUGGUUAGAGGUAGUAGGUUGCAUAGUUGAGGUAGGAGGUUGUAUAGUUGAGGUAGUAGAUUGUAUAGUUUGAGGUAGUAGUUUGUACAGUUGAGGUAGUAGUUUGUGCUGU

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miRNA Sequence Length (nts)

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miRNA Platform Overview

Purified Small RNA (Cy3)

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Direct miRNA Measurement From Total RNA

Data shown are background-subtracted signals with no filtering or normalization.

Each miRNA has signals from multiple probes.

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Purified Small RNA

miRNA Platform Overview

miRNA Profiles Using 100ng Total RNA

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Reproducibility Differential Expression

miRNA Platform Overview

Quantitative RT-PCR (qPCR) reactions (orange bars) and microarray hybridizations (blue bars) were run in quadruplicate for each miRNA species.

Microarray DataqPCR Data

Correlation between microarray and quantitative RT-PCR results

miR-34a

miR-96 miR-139

miR-141 miR-150

miR-206 miR-218

miR-335 miR-424

miR-24

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 OncologyMolecular Carcinogenesis

Nagoya University

Response from Early Access Customer

miRNA Platform Overview

miRNA Microarray System ComponentsBuilt 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:

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

YourGenomics

Story

Confident miRNAProfiling

Agilent’s DNA Microarray Platform

miRNA Platform Overview

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