The Agilent miRNA Microarray System

A New Microarray-based Tool forProfiling Human miRNAs

Choose from a comprehensive set of microarray applicationsDNARNASplice VariantsChIPElucidate the role that protein-DNA interactions play in transcription, replication, modification and repairPerform global interrogations of the transcriptome and identify alternative splice formsGXExplore gene transcription on a genome-wide basismRNAFrom one-dimensionalwith multiple applications aCGHConduct high-resolution, genome-wide profiling of DNA copy number changesCopy numberCH3Discover and monitor epigenetic modificationsMethylationTranscription FactorsmRNA isoformsmiRNAStudy microRNAs and the role they play in gene regulationmiRNA Profilingto multi-dimensional

Analysis within one contextThe Integrated Agilent Platform

miRNA Scientific Background and ImportanceDefinition: 19-30 nucleotide long single-stranded RNAs that post-transcriptionally regulate gene expressionKey regulators of gene expression, development, proliferation, differentiation, and apoptosisMay regulate >30% of human genesCurrent Sanger miRBASE Release 9.1 has 4361 entries, 474 identified in humansProjected $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)

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)

Growth of miRNA PublicationsSource: PubMed search with terms miRNA or microRNAPublication Number

The Agilent miRNA Profiling System/Workflowtotal RNA (100 ng) Direct Label with Cyanine 3-pCpHybridize on MicroarraymiRNA profilelabeled RNAData analysis withGeneSpring GX8-plex microarray

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 4 logs) Replicate probes per array (20 per miRNA) One-color analysis

Enabled by Agilent miRNA Probe Design and Direct Labeling Methodology

Direct and Sensitive miRNA ProfilingBack to Applications Platform

Probe Design1. Probe connected to array surface by stilt2. 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

Specificity to Distinguish Homologous miRNAs hsa-let-7 family

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.

miRNA Profiles Using 100ng Total RNAPlacentaPlacentaPlacentaBrainReproducibilityDifferential Expression

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

Response from Early Access CustomerWe 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 TakahashiProfessor of OncologyMolecular CarcinogenesisNagoya University

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 9.18-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:

Agilents miRNA Product Roadmap Release 1.0 May 2007Human 1.0 microarraypCp-Cy3 labeling reagentFeature Extraction 9.5Complete protocol and source information for other reagents

Release 2.0Human 2.0 microarrayMouse and Rat 1.0 microarraysLabeling kit with all required reagents

Future product releasesUpdate microarray content with discovery of new miRNAsSpike-in controlsEnabling custom miRNA contentEnhance miRNA-related features in GeneSpring GX

Questions please..Thank You!!

Several years ago, microarray analysis was focused on gene expression profiling. Today scientists are exploring multiple applications that include DNA and RNA analysis.

Agilent currently has expression (two color with one color coming Q106), aCGH to study chromosomal aberrations, ChIP-on-chip (LA) to study protein-DNA binding. When those proteins are transcription factors, LA bridges the genome, proteome and transciptome. We will also be delivering complete workflow solutions for alternative splicing, microRNA, and DNA methylation next year.

There are at least three characteristics of a microarray platform that enable it to be extended into new applications. Greater density is key because, unlike GE profiling that queries 30,000 genes, scanning in applications like aCGH and LA benefit from highest possible genome coverage and resolution. Splice variant analysis also benefits from higher numbers of features. Sensitivity is important because applications like aCGH require consistent detection of small (2x) fold changes. And in many of these new apps, samples are limited, placing a premium on lowest limit of detection. Finally, Flexibility is important because in emerging applications (e.g. microRNA, splicing) new content be incorporated into updated microarray designs on a frequent basis. Flexibility enables the kind of technology development required to perfect new applications.

Traditional method of incorporating fluorescent labels during amplification of biological sample does not apply.Limited ability to select sequences without secondary structure or with predictable Tm.Small number of targets (Hundreds, not Thousands). Exact sequences and lengths of all targets are known

Essential to array performance to keep hybridization conditions equally stringent for all tagers, this requires Tm matching, and is different for smaller miRNA sequences. We are able to accomplish this not by incorporating modified nucleotides, but by modifying probe design features.We compensate for the dye labeled nucleotide added to the probe during the labeling process, This stablizes the hybridization of the matched miRNA and its target . We adjust the size of the probe to keep it within Tm limits, according to its predicted binding sequence, (only 6-7 of the miRNA are specific for binding endogenously. To help distinguish the targeted miRNA from unintended potential targets, a hairpin loop structure is added. This destabilizes hybridization to larger nontarget RNAs, and can provide additional stabilization if the targetprobe duplex stacks with the probe hairpin.final step in our probe design strategy is empiricalselection of the optimal probe length. Hybridized with synthetic and tissue RNAs.Unmodified microarray probe (black)hybridized to miRNA target (red) is shown in (1), where the hybridizing sequence synthesized on the microarray (the probe) is connected to theglass surface by a T10 stilt (squiggly line). By adding a G on the 5 end of the probe, one more G-C pair is added to the probetarget interaction(2). When necessary, destabilization of the probetarget hybrid is achieved by eliminating base-pairing from the 5 end of the miRNA, byshortening the probe from the 3 end (3). All probes were synthesized with hairpins (4), which can increase the specificity toward thetarget miRNA and can potentially increase the stability of probetarget interactions. Agilents probes are designed with a strategy that provides both sequence and size discrimination, yielding highly specific detection of closely related mature miRNA species.One of the major challenges of miRNA array profiling is the high homologous nature of miRNA sequences. To examine the sequence specificity of our probes, 19 synthetic human miRNAs with high sequence homology to other miRNAs were individually labeled and hybridized. Very low (1 nt. Most miRNAs hybridized only to their specific probes, although among the let-7 family there is >40% cross-hybridization, hence a very difficult challenge to distinguish amongst these particular similar miRNA species.Each miRNA in the let-7 family was individually labeled and hybridized. The total signals reported by all the probes were normalized to the perfect match probetarget hybrid for each microarray. This was done using probes that were not empirically Tm matched. Specificity is expected to improve with the commericial miRNA profile array.Tight linear correlation between total RNA and small RNA of the same tissue sample. Microarray signals are specific to the small RNAs in the complex.No need for size fractionation of the total RNA from tissue samples. .

Comparison of quantitative RT-PCR (qRT-PCR) Relative expression of 10 human miRNAs was determined for seven different human tissues using both qRT-PCR (from Ambion) and our microarray assay. Results are individually plotted for each miRNA, with qRT-PCR results in orange and microarray results in blue. The miRNA levels in each tissue are reported as the fraction of the expression level in the tissue in which that miRNA is most abundant. The qRT-PCR and the microarray results agreed on which tissue expressed the highest levels of each miRNA. The qRT-PCR reactions and microarray hybridizations were each repeated four times for each miRNA. Error bars indicate one standard deviation