Post on 18-Dec-2015
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
2222222121222121
miRNA Sequence Length (nts)
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hsa-let-7 family
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
Placenta Placenta
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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