Agilent Microarrays

Kimberly Troutman
Field Applications Scientist
1-25-11
Agilent Confidential
 Agilent Microarray Platform

Overview

Applications

Comparative Genomic Hybridization (CGH)

Gene expression
 Agilent Genomics

Reagents, systems and integrated solutions enabling customers to

advance life science research

Sample Sample Sample Sample Data

Detection
Collection Prep Amp. Labeling Analysis
 Choose from a Comprehensive Set of Genomic
Applications

DNA RNA

aCGH/ CH3 ChIP

CNV
GX miRNA OLS

Copy Methylation Transcription Gene RNA Oligo library

number factors expression interference synthesis (OLS)
Cancer Research Discover and monitor Elucidate the role that
Explore gene Profile microRNAs and Gene Silencing
Cytogenetics epigenetic changes protein-DNA
transcription on a explore the role they Protein Mutagenesis
Population Studies that play a role in interaction plays in
genome-wide basis play in gene regulation Whole Gene Synthesis
cellular processes transcription,
across a variety of Genome Partitioning
replication, and repair
model systems
Santa Clara Manufacturing Facility

Industrial manufacturing Class 10,000 clean-

room
Ability to easily scale production capacity
FAB wired directly into eArray allowing direct
customer access to fully customizable
microarrays

High-performance Ink Jet Printing Enables High

Levels of Spatial Multiplexing and Flexible
Designs
Achieving High Sequence Fidelity for Oligo Library
Synthesis (OLS)
Depurination
side reaction 1) Coupling
98% cycle yield, no depurination
99.5% cycle yield, no depurination
100 98% cycle yield, with depurination
99.5% cycle yield, with depurination

3) Deblock 2) Oxidation 80

60
Repeat n times Agilent
Full Length
(%)
40
HO O Ni
O
O P O O N2 20
RO
O Conventional
O P O O N1 Processes
RO 0
O 0 50 100 150 200
O P O
RO Oligo Length
(bp)
Evolution of Ink-Jet Oligo Array Technology

2000 2004
22K 44K
135mm 100mm

55 mm 30 mm
244K 976K
2006 2009

All images 1 mm2

Agilent Array Formats
 Agilent Microarray Platform

Overview

Applications

Comparative Genomic Hybridization (CGH)

Gene expression
 Comparative Genomic Hybridization Agenda

Technical overview

Understanding CGH and CGH-SNP

What is UPD and LOH?

SNPs and copy number changes on a single array

Sample types
Understanding CGH: Comparative Genomic
Hybridization
Conventional CGH
metaphase spreads

Array CGH
BACs, cDNA's, oligos
 Understanding CGH: DNA Analytics CGH
Data Output
Heterozygous deletion (-1) Normal Amplification (>1)
Homozygous deletion (-2)
-4 -2 -1 0 +1 +2 +4
Probes
Cy5
Cy3

Red
Green
Chromosomal
Significant aberration
position
regions
Amp
Del

Exp
Notes
Ref AGW DNA Analytics module
Data shown in log2 ratio
 Understanding CGH: Analysis of Cytogenetic
Sample
Genome view

Software settings
ADM-2 threshold 10
NA14117, 46,XY,del(5)(qter>p14:).ish del(5) (D5S23-) Min 3 probes
 Understanding CGH: Analysis of Cytogenetic
Sample

Chromosome view Gene View

NA14117
46,XY,del(5)(qter>p14:)
.ish del(5) (D5S23-)

Software settings
ADM-2 threshold 10
Min 3 probes
 Choice of CGH Labeling Kits

Researchers can choose between ULS or enzymatic kits, based on their

experimental needs.

Feature Enzymatic Kit ULS Kit

Cells, frozen tissue, Cells, frozen tissue, blood,

Sample compatibility
blood degraded DNA, and FFPE

Sample requirement for

500 ng 1500 ng
1 packs
Processing time for a
4 1/2 hours 1 hour
single sample
Same for 1, 2 and 4 Per sample cost less than
pack array formats enzymatic pricing;
Cost
Cost halved for 8 Cost reduces with
packs multipacks
Breakthrough - Dual CGH+SNP Microarray Launched
September 30, 2010

Run a single microarray to detect copy number changes and copy

neutral LOH/UPD (loss of heterozygosity / Uniparental Disomy)

No need to choose between high resolution copy number data and

LOH/UPD or alternatively run two separate microarrays

Addition of SNP probes to the Agilent CGH SurePrint G3 CGH

enables the detection of blocks of LOH/UPD
CGH calls not affected by addition of SNP content
High quality of CGH calls remains
 What is UPD: Uniparental Disomy

Both members of a chromosome pair or segments of a chromosome pair

are inherited from one parent
May result in an abnormal phenotype when the chromosomes involved
are imprinted, such that only the maternal or paternal allele of the pair is
active

Remark: parental
samples needed
to detect
heterodisomy
 What is LOH: Loss of Heterozygosity

UPD in tumor cells is referred to as acquired UPD or copy neutral LOH (cnLOH)
Common in both hematologic and solid tumors

Monosomic cell LOH of arm

LOH of entire
chromosome LOH of
Normal cell
part of
arm

Interstitial
regions of
LOH

Trisomic cell Blood, Vol. 108, Issue 5, 1733-1743, September 1, 2006

SNP and Copy Number Changes: CGH and SNP
Measurements

A subset of probes on a CGH array measures SNPs, in parallel CGH probes

measure copy number, on a single array
Detection of copy neutral aberrations such as LOH and UPD
~510 Mb resolution for LOH/UPD detection across the entire genome

No change in workflow required

 SNP and Copy Number Changes: Detecting and
Measuring SNPs
Workflow Homozygous CC Heterozygous CA Homozygous AA
CG CT CG CT AG CT
CG CT AG CT AG CT
Restriction digestion
AluI : AGCT
RsaI: GTAC CG CT CG CT AG CT
CG CT AG AG CT
CT

Enzymatic labeling CG CT CG CT AG CT
CG CT AG CT AG CT

Hybridization
Wash
CG CT
CG CT

CT

CT
CG CT

AG

AG
Scan

Analyze Homozygous Heterozygous Homozygous

TWO UNCUT ONE UNCUT ZERO UNCUT
high signal intermediate signal low signal
 Supported Sample Types for CGH-SNP

Human only

Non-WGA (whole genome amplification)

Non-FFPE

Non-cancer

Challenges associated with cancer samples

Normal clone contamination
Subclones (i.e., tumor heterogeneity0
Copy number: 0, 1, 2, 3, 4, 5
Samples not necessarily mostly diploid
Some cancer samples only available as FFPE tissue
Algorithm development for cancer ongoing
 Agilent Microarray Platform

Overview

Applications

Comparative Genomic Hybridization (CGH)

Gene expression
 Low Input Quick 4x44K v2 SurePrint G3 Additional
Amp New Higher Content
Smaller content throughput Exon
sample Lower price arrays
Lower price

Introducing
Low Input Quick Amp Labeling Kit (LIQA)

Equivalent performance with smaller starting sample requirements and lower

price per sample
 Overview of Protocol

AffinityScript Start with 25 ng total RNA

Create dsDNA containing T7 promoter:
AffinityScript, RT with high efficiency
Oligo-dT primed
Create labeled cRNA (anti-sense)
T7 RNA polymerase
Cy3-CTP (Cy5-CTP)
Linear amplification IVT
Purified cRNA is labeled ready for hyb

Linear amplification to avoid

introducing unnecessary biases
Single-tube reaction, no cDNA clean-
up step for fast and easy processing
 Product Overview: LIQA Features & Benefits
Feature
10 ng input
Lower cost
Linear amplification labeling
Fewer tubes, reagents
blended
No cDNA clean-up
~ 5 logs dynamic range
Reproducibility and
accuracy
Compatible with eukaryote
designs and formats
Benefit
Preserve precious material! Small samples amount
Run more samples or reserve money for downstream
applications
Accurately represent the prevalence of RNA transcripts
during labeling, avoid introducing unnecessary biases
Easier to use assay, decreased pipetting, no need to pipette
the viscous PEG
Process quickly and simply with single-tube chemistry
Detect the low expressers (high ones, too!)
More accurately represent range of gene expression in the
sample
Feel confident in the results
Use the same kit and assay for all study designs
Agilent Confidential
 Low Input 4x44K v2 SurePrint Additional
Quick Amp G3 Content
New
Smaller sample content Higher
Exon
arrays
Lower price throughput
Lower
price

Introducing
4x44K V2 Gene Expression Arrays
Product Overview: Introducing 4x44K v2 Arrays for
Human, Mouse, & Rat
With current content, based on updated transcriptome databases
Full coverage of human, mouse, and rat genomes
All standard controls included, plus probes for 12 ERCC controls
Providing consistent, high quality data
Wide dynamic range (~ 5 logs)
High reproducibility (< 15% CV between arrays or slides)
For confidence in gene expression results

Human Mouse Rat

Old New Old New Old New
% mRNAs
covered
74.6 % 96.8 % 56.0 % 79.8 % 61.5 % 87.7 %

Total unique
genes covered
19,596 27,958 21,503 34,017 17,366 26,930
 Product Overview: 4x44K v2 Features & Benefits
Feature
Updated content for human,
mouse, and rat
Same performance as v1 arrays
Wide dynamic range
High reproducibility, accuracy
Compatible with LIQA
Low RNA input
Single-tube chemistry
Annotations updated regularly
Benefit
Comprehensive coverage based on current
databases
Detect low expressors and high expressors
More accurately represent range of
expression levels
Feel confident in the results
Preserve precious material
Work with smaller samples
Easy, simple assay
Decreased pipetting, fewer opportunities for
errors
Annotations stay current with evolving
databases
 Low Input Quick 4x44K v2 SurePrint G3 Exon
Amp New Higher Exon
Smaller content throughput analysis
sample Lower price
Lower price

SurePrint G3
8x60K Gene Expression Arrays
Product Overview: Product Structure for SurePrint
G3 Gene Expression Arrays
8x60K prices are set according to whole-genome coverage
Price advantages for 8x60K can be reached via bundling with LIQA or
volume purchases
# Slides
Part Number Microarray
(# Arrays)
G4851A SurePrint G3 Human GE 8x60K Kit 3 (24)
G4852A SurePrint G3 Mouse GE 8x60K Kit 3 (24)
G4853A SurePrint G3 Rat GE 8x60K Kit 3 (24)
G4102A SurePrint G3 Custom GE 8x60K 1 (8)
G4862A SurePrint G3 Custom GE 4x180K 1 (4)
G4861A SurePrint G3 Custom GE 2x400K 1 (2)
G4860A SurePrint G3 Custom GE 1x1M 1 (1)
G4858A SurePrint G3 Unrestricted GE 8x60K 1 (8)
G4857A SurePrint G3 Unrestricted GE 4x180K 1 (4)
G4856A SurePrint G3 Unrestricted GE 2x400K 1 (2)
G4855A SurePrint G3 Unrestricted GE 1x1M 1 (1)

Agilent Confidential
 Product Overview: SurePrint G3 Catalog Arrays

General
8x60K format: human, mouse, rat (more may follow)
10 ng for 8x60K & 8x15K formats and 25 ng for 4x formats
Content
Biological same as 4x44K v2
Human array will also include lincRNAs
Controls same as 4x44K v2, except
4x44K v2 arrays include 12 ERCC probes, while
8x60K arrays will include all 96 ERCC probes

Use of additional space on arrays

Replicate probes
For multiplicative detrending use 10x
Further filling to 3x
Reserved for added biological content, such as linc RNAs (mouse)
Probe sets are available on eArray for custom design and additional content
 Product Overview: G3 Enables Inclusion of
Additional LINCs RNA Content
Non-coding RNAs: functional RNAs not translated into proteins
miRNA are an example of non-coding RNAs
Long intergenic non-coding (linc) RNAs
>200nt, most are 1 kb to 10 kb
Poly-adenylated, spliced
Compatible with LIQA-based labeling
>1K lincRNAs identified by Guttman M et al

Included on human and mouse catalog designs (no plans for rat)
Product Overview: SurePrint G3 Features & Benefits
Feature
Increased content
(human lincs, custom)
Higher throughput
More affordable
Higher density
Updated content for human, mouse,
and rat
Same performance as HD arrays
Wide dynamic range
High reproducibility, accuracy
Compatible with LIQA
Low RNA input (>10 ng)
Single-tube chemistry
Annotations updated regularly
Benefit
More comprehensive coverage, ability to correlate
lincRNA and mRNA expression
Easier lab processing
Run more samples to increase power of a gene
expression study
More flexibility in designs: look at splicing events or
multiple species on an array (eg, human +
bacteria)
Comprehensive coverage based on current
databases
Detect low expressors and high expressors
More accurately represent range of expression
levels
Feel confident in the results
Preserve precious material
Work with smaller samples
Easy, simple assay
Decreased pipetting, fewer pipetting errors
Annotations stay current with evolving databases
Low Input 4x44K 8x60K Additional
Quick Amp Redesign Higher Content
Smaller sample New throughput Exon
Lower price content Lower price arrays

Exon Arrays
There here!
Agilent Gene Expression Exon Arrays
 Catalog and Custom Arrays

Wide range of catalog microarrays available

Human, mouse, rat, bovine, C.elegans, canine, chicken, drosophila, horse,
mosquito, porcine, rabbit, rhesus macaque, salmon, sheep, xenopus, zebrafish
Arabidopsis, barley, brassica, cotton, maize, medicago, rice, tobacco, tomato, wheat
E. coli, magnaporthe, yeast
Unlimited custom microarray options
Choose Agilent-optimized probes, upload your own sequences, or design probes
using eArray tools
Search and download any Agilent catalog microarray content with free access and
use in your designs
 Model Organism Gene Expression Arrays

Over 28 catalog array designs

Collection of arrays growing rapidly

Annotations updated regularly

Content can be customized in eArray

 Any Array or Genome

The number of sequenced genomes

is on the climb
Agilents gene expression solution
offers custom arrays for unlimited
species
Upload probes for any sequence
Visit eArray to design any custom
microarray
https://earray.chem.agilent.com/earray/

Genome Projects: H. sapiens, H. neanderthalensis, H. influenzae, M. musculus, R. norvegicus, P. troglodytes, M. mulatta, G. gallus, M. eugenii, F. silvestris,
C. lupus familiaris, D. melangaster, S. cerevisiae, S. pombe, N. crassa, A. thaliana, O. sativa, T. aestivum, Z. mays, P. trichocarpa, E. coli, SARS virus, S.
purpuratus, C. elegans, B. rerio, X. laevis, O. latipes, T. rubipres, S. lycopersicum, S. tuberosum, A. mellifera, V. vineifera, O. anatinus, A. pisum, etc.
 Agilent Microarrays

Most flexible commercial microarray platform

Leader in CGH/CNV array analysis

Extensive gene expression array catalog of >25 genomes

Complete solution from sample preparation through data

analysis