Sensitive Gene Expression Using Minimal RNA Amounts · PDF fileSensitive Gene Expression Using...

Sensitive Gene Expression Sensitive Gene Expression Using Minimal RNA Amounts

Introducing Agilent’s SurePrint G3 Gene Expression PlatformGene Expression Platform

Stephanie Fulmer-Smentek, Ph.DRNA Applications Manager, Genomics R&D

TopicsThe Agilent Gene Expression PlatformThe Agilent Gene Expression Platform

Introducing the SurePrint G3 Gene Expression Microarrays

Low Input Quick Amp Labeling

The Agilent Gene Expression PlatformThe Agilent Gene Expression Platform

Quality from beginning to end

A Complete Solution for Gene ExpressionRNA ifi ti• RNA purification

• RNA quality assessment

• Catalog arrays for 30

qRT-PCR Validation

Data Analysis

a a og a ay o 30species

• Unlimited custom array options

Array Selection & Creation

Scanning and Feature Extraction

p

• Simple assay with low RNA input requirement

• Quality controls

Genome wide &

Feature Extraction • Quality controls

• Biological interpretation

• qRT-PCR validation

Sample Labeling

Genome-wide & Custom ArraysHyb &

Wash

Sample Prep and QA

Catalog and Custom Arrays through eArray• Wide range of catalog microarrays availableg g y

– 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 li M h Y– E. coli, Magnaporthe, Yeast

• Unlimited custom microarray options in eArray

– Choose Agilent-optimized probes, upload your own sequences, or design probes

S h d d l d A il t t l i t t d i l d i d i– Search and download Agilent catalog microarray content and include in your designs

ORANGE: Catalog arrays available in SurePrint G3 8x60K format

Any Array, Any Genome

• The number of sequenced genomes is climbing

• Agilent’s gene expression solution offers custom arrays for unlimited species

l d d b f• Upload or design probes for any sequence

• Visit eArray to design any t icustom 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 l f ili i D l t S i i S b N A th li O ti T ti Z P t i h E li SARS i SC. 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.

Wide Dynamic Range for Sensitivity to Low Expressors

• Agilent gene expression data provides the expected normal distribution of • Agilent gene expression data provides the expected normal distribution of signals, with few probes in the noise

• Competing platforms suffer from compressed signals without a normal distribution

Sensitive Microarray-based Gene Expression • All Agilent microarrays include probes for spike-in controls, which g y p p ,

indicate labeling and hybridization success

• Agilent spike-ins demonstrate

• Sensitive detection on the order of 1 in 6 9 million transcripts• Sensitive detection on the order of 1 in 6.9 million transcripts

• Detection across a dynamic range spanning 5 orders of magnitude

Quality Controls and Quality Metrics are an Integral Part of the Agilent Gene Expression Platform

• Spike in kits to track labeling efficiency and success• Spike-in kits to track labeling efficiency and success

• QC Report with metrics on signal, background, reproducibility, and spike-ins

• Histogram of signals• Histogram of signals

• And much, much more!

Introducing theIntroducing theNew SurePrint G3 Gene Expression

MicroarraysMicroarrays

More comprehensive coverageLower RNA input

Continued confidence in results

Introducing

Agilent Gene Expression Microarray FormatsIntroducing

SurePrint G3 ArraysSurePrint HD Arrays

1x 44K 1M44K 244K

2x 44K400K44K105K

4x 44K180K44K44K

8x60K15K

65 µm features

Compatible with:Agilent B or C Scanner

30 µm features

Compatible with:Agilent C Scanner

For research use only. Not for use in diagnostic procedures

SurePrint G3 GE Array OfferingCatalog arrays on 8x60K formatsg y

• Comprehensive coverage– Updated content

– Inclusion of noncoding RNAs for the first time!– Inclusion of noncoding RNAs, for the first time!

• Higher throughput for whole-genome studies

• Lower RNA input requirement (down to 10 ng)

I d ff d bilit• Increased affordability

Unlimited custom options

• Available in 8x60K, 4x180K, 2x400K, and 1x1M

• Added density makes it possible to– Combine species on a single array; for example human + pathogensCombine species on a single array; for example, human + pathogens

– Target exons or splice junctions

– Replicate probes


SurePrint G3 8x60K Microarrays Arrays:Compared to the Original 4x44K Arrays for Human, Mouse, and Rat

New:

• New content: more relevant and comprehensive coverage, based on updated transcriptome databasestranscriptome databases

• Includes lincRNA probes (human and mouse only)

• Includes 96 ERCC* control probes

Continued:

• Based on RefSeq, Ensembl, Unigene, and GenBank mRNA

• Transcript-based designs• Transcript-based designs

• High-quality, high-standard 60-mer probes

• Reliable gene expression data

• Annotations updated regularlyAnnotations updated regularly

• All standard controls included

*External RNA Controls Consortium


Agilent Gene Expression Catalog Array Content

EntrezSpecies Array Format

Entrezgene

targets

lincRNAtargets

Spike-in controls

Databases used for design

SurePrint G3 8x60K 27,958 7,419 10 Agilent

96 ERCCRefseq Build 36.3;

Human8x60K , , 96 ERCC

qEnsembl Release 52; Unigene Build 216 (Apr 2009);GenBank mRNA (Apr 2009)

SurePrint HD 4x44K v2 27,958 Ø 10 Agilent

12 ERCC

SurePrint G3 10 Agilent

Mouse

SurePrint G3 8x60K 34,017 4,623 10 Agilent

96 ERCCRefseq Build 37;Ensembl Release 55;Unigene Build 176 (Apr 2009);GenBank mRNA (Apr 2009)


12 ERCC

Rat

SurePrint G3 8x60K 26,930 Ø 10 Agilent

96 ERCCRefseq Build 36.2;Ensembl Release 55;Unigene Build 177 (Oct 2008);GenBank mRNA (Jan 2009)


12 ERCC4x44K v2 12 ERCC

• SurePrint G3 catalog arrays include the same mRNA probes as SurePrint HD 4x44K v2 microarrays

• SurePrint G3 catalog arrays also include lincRNA probes for human and mouse


G3 Density Enables Inclusion of lincRNA Probes• Long intergenic non-coding (linc) RNAs g g g ( )

• > 200 nt, most are 1 kb to 10 kb

• Poly-adenylated, spliced

• Compatible with LIQA-based labelingp Q g

• Probes selected for human and mouse lincRNAs• Via collaboration with John Rinn (Broad Institute)

• Collaboration is ongoing: further lincRNA identification, refining probe selectiong g , g p

• This represents the first commercial microarray including both coding and non-coding targets

Nature 458, 223-227 (1 February 2009)


Nature Biotechnology 28, 503-510 (2 May 2010)

Including lincRNAs on Gene Expression MicroarraysWhat they are, and why they are relevant.

• Non-coding RNA genes include tRNA, rRNA, snoRNA, miRNA, siRNA, piRNA, lincRNA, …

• lincRNAs perform important biological • lincRNAs perform important biological functions

• There are ~ 10-15 well-characterized lincRNAs in mammals, with distinctive b l l l h h d l lbiological roles through diverse molecular mechanisms:

– X-chromosome inactivation (Xist, Tsix)

Imprinting (H19 Air)– Imprinting (H19, Air)

– Trans-acting gene regulation (HOTAIR)

– Regulation of nuclear import (Nron)

http://www.rikenresearch.riken.jp/eng/frontline/5920

• Some studies implicate lincRNAs in disease states and suggest involvement cancer


Introducing theIntroducing theNew SurePrint G3 Gene Expression

MicroarraysMicroarrays

THE DATA

SurePrint G3 Microarrays for High ReproducibilitySurePrint G3 8x60KSurePrint G3 8x60K SurePrint G3 8x60K

10 ng inputLog2 Ratios, 2-color

SurePrint G3 8x60K10 ng input

Log2 Signals, 1-color

• Signals and log ratios are highly correlative across technical replicates for both 1-color and 2-color experimentsp p


High Reproducibility Across SurePrint G3 Arrays• High inter-array reproducibility indicated by low % coefficient of g y p y y

variance between replicate probes on individual arrays

• SurePrint G3 arrays provide data you can trust

ted

nes

Dete

ct

CV

(%

)

# G

en


SurePrint G3 Arrays Continue High Concordance and Correlation to TaqMan Data

Agilent Array Correlation to Taqman

y Lo

g R

atio

Mic

roar

ray

10 ng Input: n = 686 r = 0 93 m = 0 86

TaqMan Log Ratio

10 ng Input: n 686, r 0.93, m 0.86 50 ng Input: n = 704, r = 0.94, m = 0.90

• As compared with other microarrays providers, Agilent array gene expression signals span a wider dynamic range

• With wide dynamic range, Agilent gene expression data reflect real gene de dy a c a ge, g e ge e e p ess o da a e ec ea ge eexpression levels


Agilent Gene Expression for Reliable Profiling An array is not just an arrayy j y

Agilent arrays provide higher reproducibility across a wider dynamic range

Agilent arrays provide sensitivity to genes of low and high abundance

Agilent 4x44KMAQC A Sample

Nimblegen 12x135KMAQC A Sample

15 15R = 0.997 R = 0 963R = 0 993

Agilent 8x60KMAQC A Sample

15

5

10

5

10

R = 0.99725 ng

R = 0.96310 µg

R = 0.99310 ng

5

10

-5

0

-5

0

-5

0

-5 0 5 10 15-5 0 5 10 15 -5 0 5 10 15

Agilent data: Log2 75th percentile-normalized signals


g e t data og2 5 pe ce t e o a ed s g a sNimblegen data: Log2 quantile normalized signals

Agilent Microarrays On Par with DGE, High Correlation to qRT-PCRAgilent array QC:

• Median %CV of replicate probes < 7

• Pearson correlation (R) across technical replicates > 0.99

Illumina RNA-Seq QC:

• % CVe of replicated DGE measurements < 9

• Pearson correlation (R) across technical replicates > 0.99

Agilent arrays, RNA-Seq, qRT-PCR all correlate


Feng et al., Mol Biotechnol 2010

Agilent Microarray: 4x44K, Agilent Scanner, GeneSpringRNA-Seq: Illumina Genome AnalyzerqRT-PCR: Mx3005P QPCR System

SurePrint G3 Microarrays and RNA-Seq: a Comparison

St ong co elation of log atios a e • Strong correlation of log ratios are observed between microarray and RNA-Seq for samples MAQC A and B

• Microarrays are faster easier more M = 1.120

Array and RNA-SeqLog2 Ratios (B/A)

• Microarrays are faster, easier, more affordable, and require little RNA input

R = 0.840N = 15516

µg

Agilent G3 LIQA RNA-Seq

A-S

eq,

1 +LIQA RNA Seq

Starting Material 10 ng 1 µg

Total Sample Prep 6.5 hr 11.5 hr

RN Hands-on Sample

Prep 1.5 hr 3.75 hr

Sample to Data 1.5 days ~ 1 week

8x60K, 25 ng, 1-colorAnalysis Simple Challenging

Price per sample $$ $$$


The Low Input Quick Amp Labeling KitThe Low Input Quick Amp Labeling Kit

For sensitive gene expressionusing low RNA input amounts

The New Low Input Quick Amp Labeling Kit (LIQA):Comparison to Quick Amp

Quick Amp LIQA Advantage13 tubes 10 tubes, including H2O Easier to use

MMLV-RT Stratagene AffinityScript-RTHigher labeling efficiency: more yield from smaller samplessamples

Input range:1-color: 200 ng to 1 µg2-color: 50 ng to 2 µg

Input range:1-color: 10 ng to 200 ng2-color: 10 ng to 200 ng

Less RNA required,more RNA preserved

20 reactions per kit 24 reactions per 1-color kit48 reactions per 2-color kit

Compatible with 8x format

Higher volumes, more reagents higher cost

Lower volumes, fewer reagents lower cost Price savingsreagents higher cost reagents lower cost

Customers are NOT advised to switch kits in the middle of an experimentCustomers are NOT advised to switch kits in the middle of an experiment


LIQA Works on G3 Arrays Across Broad Range of Inputs

MAQC A MAQC B

5-50ng 5-15ng 25-100ng100-500ng 200-500ng

• Only 10 ng of total RNA required for 8x formats

• Only 25 ng of total RNA required for 4x formats

• 25 ng is the recommended input amount for both25 ng is the recommended input amount for both


Inter-array %CV Comparisons and # of Detected Genes

• Data is highly reproducible between arrays• Data is highly reproducible between arrays

40 40000 N

25

30

35

) 25000

30000

35000

umber of

15

20

25

CV (%

)

15000

20000

25000

f Genes D

5

10

5000

10000

Detected

0

10 15 25 50 100

200

500 10 15 25 50 100

200

500

0

MAQC A Input (ng) MAQC B Input (ng)MAQC_A Input (ng) MAQC_B Input (ng)


Summary• Agilent Gene Expression microarrays provide the highest quality data

– Sensitivity to detect low expressors and identify small fold changes– Correlation with Taqman and qRT-PCR– Unlimited custom options– Comprehensive catalog products

• Agilent introduces the New SurePrint G3 Gene Expression Microarraysi i (10 )– Lowest RNA input requirement (10 ng)

– Comprehensive coverage including coding and non-coding RNAs– Higher density and throughput, same performance

• Agilent introduces the Low Input Quick Amp Labeling Kit– Consistent labeling with both one-color and two-color options– Compatible with SurePrint G3 and SurePrint HD microarrays– Compatible with SurePrint G3 and SurePrint HD microarrays

• The Agilent Gene Expression Platform brings quality results from RNA isolation through microarray processing and qRT-PCR validation


QUESTIONS?Thank you

QUESTIONS?

Overview of ProtocolWorkflow diagram for the Low Input Quick Amp

AffinityScript

Workflow diagram for the Low Input Quick Amp Labeling Kit:

– 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

AffinityScript

–T7 RNA polymerase

–Cy3-CTP (Cy5-CTP)

–Linear amplification IVT

– Purify cRNA labeled product ready for hyb

Linear amplification to avoid introducing unnecessary biasesg ySingle-tube reaction, no cDNAclean-up step for fast and easy processing


Microarray Performance Benefits from Long Oligos

Sensitivity Specificity

• 60-mer oligos provide the strongest sensitivity and the strongest specificity

• Agilent SurePrint technology enables the highest of quality for long oligos

Hughes et al., Nature Biotechnology 2001

The Agilent Microarray Scanner Enables Dynamic Range for Accurate Gene Expression Detection

Alternate ScannerAgilent B Scanner

(5 µm resolution)Compatible with HD Arrays

Agilent C Scanner(2 µm resolution)

Compatible with G3 & HD Arrays

16 bit single scanDynamic Range – 4 logs

16 bit and XDR scanDynamic Range – 5 logs

20bit single scanDynamic Range – 5 logs

10 102 103 104 105 2 3 4 5 6 2 3 4 5 610 102 103 104 105 10 102 103 104 105106 10 102 103 104 105106

News Flash!

Probe Designs for SurePrint G3 8x60K Microarrays:A Comparison to the Original 4x44K Microarrays

Human Mouse Rat

T l # P b f ETotal # Probes for EntrezGene Targets 34,127 39,429 30,367

% Probes Retained from 54 4% 36 6% 38 2%% Probes Retained from the Original 4x44K Arrays 54.4% 36.6% 38.2%

Old probes were replaced with new probes when

• Old probes were no longer relevant, based on current databases

• New probes gave better scores based on updated probe scoring algorithms• New probes gave better scores, based on updated probe scoring algorithms


SurePrint G3 Metrics Consistently Indicate Reliability• High intra-array reproducibility indicated by low % coefficient of g y p y y

variance between replicate probes on individual arrays

• SurePrint G3 arrays provide data you can trustnal

10

MAQC AMAQC B

cess

ed S

ign

8

% C

V P

roc

6

1x1M 2x400K 4x180K 8x60K

4


Replicate Arrays Indicate High Reproducibility for Multiple Sample Sources • Reproducibility is equivalent for 4x44K and 8x60K formats

• Reproducibility is equivalent for gene probes and lincRNA probes

40 4000040 40000

25762

34617

33686

36790

3567134912

30086

2817726130

2701126061 27096

28177

25

30

35

40

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30000

35000

40000

Num

berNum

ber25762

34617

33686

36790

3567134912

30086

2817726130

2701126061 27096

28177

25

30

35

40

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30000

35000

40000

Num

berNum

ber25762 2613025466 25116

15

20

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CV (%

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15000

20000

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r of Genes D

eter of G

enes Dete

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20

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CV (%

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enes Dete

0

5

10

07 16 RR ES RA 07 16 RR ES RA 07 16 RR ES RA

0

5000

10000

ectedected

0

5

10

07 16 RR ES RA 07 16 RR ES RA 07 16 RR ES RA

0

5000

10000

ectedected

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

NPC

‐10

NPC

‐11

UMR

mE

mES‐R

4x44K 8x60KProtein-coding +

Protein-coding Genes OnlyProtein-coding +

lincRNAs


lincRNA Probes Function and Perform Like mRNA Probes • High reproducibility observed for all probes across 5 logs of dynamic range

• Mouse ES cell RNA applied in replicate to SurePrint G3 Mouse 8x60K Microarrays

All Biological Probes on 8x60K Array lincRNA Probes Colored GreenGreenAll Biological Probes on 8x60K Array lincRNA Probes Colored GreenGreen

ange

g y G ee

ange

g y G ee

ES Rep

licate #3

ear Dynam

ic Ra

ES Rep

licate #3

ES Rep

licate #3

ear Dynam

ic Ra

ES Rep

licate #3

All Biological Probesr = 0 997

Log 2

mE

Five Logs Line

Log 2

mE

lincRNA Probes Onlyr = 0 995

All Biological Probesr = 0 997

Log 2

mE

Five Logs Line

Log 2

mE

lincRNA Probes Onlyr = 0 995r = 0.997

n = 34,599

Log2 mES Replicate #1 Log2 mES Replicate #1

r = 0.995n = 8,947

r = 0.997n = 34,599

Log2 mES Replicate #1 Log2 mES Replicate #1

r = 0.995n = 8,947


Tissue-specific Expression of lincRNAs Demonstrated• Several lincRNAs are specifically expressed in the CNS during embryonic

development (Guttman et al., 2009)

• Brain-specific expression consistent with array results across multiple cell lines, including expression in mES cells only when treated with retinoic acid

lincRNA‐1 lincRNA‐2

Brain‐Specific

lincRNA‐1 lincRNA‐2

Brain‐SpecificmRNAmRNA

8x60K Microarray Data Visualized with GeneSpring GX 11.0

ls8x60K Microarray Data

Visualized with GeneSpring GX 11.0

lsqPCR Data lincRNA 1

lincRNA‐1

Brain‐Specific mRNA

lincRNA‐2Microarray Signa

lincRNA‐1

Brain‐Specific mRNA

lincRNA‐2Microarray SignalincRNA‐1

lincRNA‐2

lincRNA‐2

Log 2

*

lincRNA‐2

Log 2

*


Unpublished data courtesy of Guttman et al.

Signals Are Reproducible Across Wide Dynamic Range for Tumor and Normal Samples

of m

agni

tude

icat

e #1

cate

#1

~5 o

rder

s

Replicate #2

Rep

li

Replicate #2

Rep

lic

AdenocarcinomaNormal Colon

Rep

licat

e #2

Rep

licat

e #

2

• Various tissue samples and types provide similar levels of

Replicate #3 Replicate #3

R

p yp phigh reproducibility using SurePrint G3 Gene Expression Microarrays

• Reproducibility translates in the ability to confidently identify genes of differential expressiongenes of differential expression


Technical Reproducibility for Various MicroarraysNimblegen

1 385KNimblegen Agilent

R= 0.999510 ng

R= 0.963010 µg

R= 0.989810 µg

1x385K 12x135K 8x60K

14 14 10

Replicate 2

8

10

12

8

10

12

5

6 8 10 12 14

6

8

6 8 10 12 14

6

8

0 5 10

0Dynamic Range = 3.3

Dynamic Range = 3.3

Dynamic Range = 4.5

SurePrint G3 Gene Expression Arrays provide:

Replicate 1 Replicate 1 Replicate 16 8 10 12 14 6 8 10 12 14 0 5 10

p y p

• Better correlation

• Wider dynamic range

• Access to smaller RNA samples


Agilent Microarrays Perform On Par with DGEArray and NGS are both reproducible and correlative, with a slope of ~ 1y p , p

OPD = log2 [microarray signal (sample A) * DGE signal (sample B)]


Feng et al., Mol Biotechnol 2010OPV = log2 [microarray signal (sample B) * DGE signal (sample A)]OPD & OPV were fitted to a regression line that was very close to the ideal 45 line

Features & Benefits: LIQA

Feature Benefit

10 ng input Preserve precious material! Work with small samples!

Lower cost Run more samples or reserve money for downstream applicationsLower cost Run more samples or reserve money for downstream applications

Linear amplification labeling

Accurately represent the prevalence of RNA transcripts during labeling, avoid introducing unnecessary biases

Fewer tubes, Easier to use assay, decreased pipetting, no need to pipette the reagents blended viscous PEG

No cDNA clean-up Process samples quickly and simply using single-tube chemistry

~ 5 logs dynamic Detect the low expressors (and the high ones, too!)M l f i i h lrange More accurately represent range of gene expression in the sample

High reproducibility, accuracy

Feel confident in the results

Compatible with all Compatible with all eukaryote designs

and formatsUse the same kit and assay for all study designs


Comparisons of LIQA and Quick Amp to TaqMan Data

L I t Q i k AQ i k A

Rat

io

Low Input Quick AmpQuick Amp

roar

ray

Log

TaqMan Log Ratio TaqMan Log Ratio

Mic

r

• Agilent GE microarrays show the highest correlation to TaqMan data

TaqMan Log Ratio TaqMan Log Ratio10 ng Input: n = 686, r = 0.93, m = 0.86 50 ng Input: n = 704, r = 0.94, m = 0.90

50 ng Input: n = 661, r = 0.92, m = 0.91 200 ng Input: n = 691, r = 0.93, m = 0.92

• Agilent GE microarrays show the highest correlation to TaqMan data

• This remains true using the Low Input Quick Amp Labeling kit

• LIQA provides the added advantage of an increase in the number of genes detected without any compromises detected, without any compromises


Features & Benefits: SurePrint G3 Gene ExpressionFeature Benefit Feature Benefit

• Comprehensive coverage

• Sensitivity to known genes according to recent databases

• Ability to correlate lincRNA and mRNA expression in a single experiment

U d t d t t f h C h i b d t • Updated content for human, mouse, and rat

• Comprehensive coverage based on current databases

• Higher density • Increased flexibility in designs: splicing events, multiple species on an array, etc

• Detect low expressors and high expressors• Same performance as HD arrayso Wide dynamic rangeo High reproducibility, accuracy

• Detect low expressors and high expressors• More accurately represent range of

expression levels• Feel confident in the results

• Annotations updated regularly • Annotations stay current with evolving d t b Annotations updated regularly databases

• Higher throughput for whole-genome • Easier lab processing

• Compatible with LIQAL RNA i t (d t 10 )

• Preserve precious material• Work with smaller samples

E i l o Low RNA input (down to 10 ng)o Single-tube chemistry

• Easy, simple assay• Decreased pipetting, fewer opportunities for

errors

• More affordable • Run more samples, increase powerp , p

Agilent Confidential

Inter-array Reproducibility and # of Detected Genes

3332533902

3322334051

3508535000

40000

35

40N

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Low Input Quick AmpQuick Amp

3151230977

3332531892

34051

20000

25000

30000

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CV (%

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10000

15000

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0

5000

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A B A B A B A B

s

50 ng 200 ng 10 ng 50 ng

• The new LIQA kit results in lower CVs and more genes detected

50 ng 200 ng 10 ng 50 ng


LIQA Enables Low Input Labeling for Microarray Analysis • RNA input requirement as low as 10 ng for 8x formatsp q g

(25 ng recommended)

4x formats

8x formats

Yield Requirements

formats formats

1-color 1.65 µg 600 ng

2-color 825 ng 300 ng


Agilent Arrays Provide Accurate Gene Expression Values

• AffinityScript QPCR cDNA Synthesis Kit

Brilliant III Ultra Fast QPCR Master Mix• Brilliant III Ultra-Fast QPCR Master Mix


Assessment of Cross-contamination:Visualization of Array Images in Various Color Channels(example from customer data)(example from customer data)

BothC lColor

Channels

GreenChannel

OnlyOnly

RedChannel

Only

One-Color Intra-array Median %CVs for Replicated Biological Probes( l f t d t )(example from customer data)

4

5

6

d Si

gnal

s

4

5

6

d Si

gnal

s

1

1

2

3

%C

V Pr

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sed

251503410073 251503410087

1 2 3 4

12

01 2

34

1

2

0

251503410073 251503410087

Lime Green = MAQC_Sample_ADark Green = MAQC_Sample_B

One-Color Inter-array Median %CVs for Normalized Biological Probes( l f t d t )(example from customer data)

4

5

Vs (n

=4)

1

2

3

cros

s Sl

ide

%C

V

0

1Ac

A B

Across Arrays and Slides (n=4)Across Arrays and Slides (n 4)

Lime Green = MAQC_Sample_ADark Green = MAQC_Sample_B

The advantage of 5 log dynamic range

• 5 log Dynamic Range allow detect both high and low

MCF-7GO analysis for each Expression range

abundant transcripts

MetabolismBiosynthesis

Regulation of transcriptionRegulation of transcription

Signal transduction

Pa

Features & Benefits: 4x44K v2

Feature Benefit Feature Benefit • Updated content for human,

mouse, and rat• Comprehensive coverage based on current

databases

• Same performance as v1 arrayso Wide dynamic rangeo High reproducibility,

accuracy

• Detect low expressors and high expressors• More accurately represent range of

expression levels• Feel confident in the resultsy

• Compatible with LIQAo Low RNA input

• Preserve precious material• Work with smaller samples• Easy simple assayo Low RNA input

o Single-tube chemistry• Easy, simple assay• Decreased pipetting, fewer opportunities

for errors

• Annotations stay current with evolving • Annotations updated regularly • Annotations stay current with evolving databases

4x44K v2 Microarrays: Standard Agilent Quality• High reproducibility Technical Replicates, 4x44K v2g p y

and reliability R = 0.997 R = 0.997 R = 0.996

• With wide dynamic range, Agilent gene expression arrays

Dynamic Range (Orders of Magnitude)

6Human Mouse Rat

continue to reflect real gene expression levels

3

4

5

1

2

3

4x44K4x44K v2

Kit Comparison: Hierarchical Clustering

S l A S l BSample A Sample B

10ng 50ng 200ng 50ng 10ng 50ng 200ng 50ngLIQA QA LIQA QA

g R

NA

Inpu

t 10ng 50ng 200ng 50ng 10ng 50ng 200ng 50ng

Kit

–20

0 ng

Qui

ck A

mp


LIQA: Fewer Tubes for Easier Sample ProcessingQuick Amp LIQAQuick Amp LIQAT7 Promoter Primer T7 Promoter Primer

Mix

5X First Strand Buffer 5X First Strand Buffer

0 1 M DTT 0 1 M DTT

cDN

AM

ast

er

M 0.1 M DTT 0.1 M DTT

10 mM dNTP Mix 10 mM dNTP Mix

MMLV-RTAffinityScript RNase Block Mix

RNaseOUTRNaseOUT

Nuclease-free water (not included) Nuclease-free water

4X Transcription Buffer5X Transcription Buffer

50% PEG

nsc

rip

tio

n

ast

er

Mix

50% PEG

0.1M DTT 0.1M DTT

NTP mix NTP mix

RN OUT

Tra

nM

a RNaseOUT

T7 RNA Polymerase BlendInorganic pyrophosphytase

T7 RNA Polymerase

C i 3 CTP ( C i 5 CTP) C i 3 CTP ( C i 5 CTP)


Cyanine 3-CTP (or Cyanine 5-CTP) Cyanine 3-CTP (or Cyanine 5-CTP)

Absolutely RNA Purification Kits Provide Quality RNA for Downstream Applications

• High yields of highly pure total RNA

• Fast 30 minute method

• Non-toxic

• DNase included for gDNA removal

• Convenient bench-top storage saves freezer space

A plethora of options:

• Absolutely RNA Miniprep Kit (cells, tissues)

• Absolutely RNA Microprep Kit (cells, 96-well version available)

• Absolutely RNA Nanoprep Kit (smallest samples, down to single cell)

• Absolutely RNA miRNA Kit (for total RNA containing miRNA)

• Absolutely RNA FFPE Kit (with and without deparaffinization reagents)

Bioanalyzer results of cell RNA purified using the Absolutely RNA Miniprep Kit

qRT-PCR Solutions for Accurate and Reproducible Microarray Validation MX3005P

• Agilent Stratagene offers a wide portfolio of qPCR reagents featuring the Brilliant III qRT-PCR master mixes

• The MX3000P and MX3005P, real-time thermal cyclers,

3005

ycombine high performance with full features and flexibility

37.5

Brilliant II SYBR Green qRT-PCR 1-Step Kits versus competitors

34.5

35.5

36.5

ge

31.5

32.5

33.5

Ct

ran

g

28.5

29.5

30.5

Brilliant II Company A Company I Company Q Brilliant I

8 replicates at 2 pg total RNA; ARF target

p y p y

SurePrint G3 Arrays are Sensitive to Small ChangesMicroarray/qRT-PCR correlation is > 0.96 for genes with fold change < 1.3y/q g g

Microarray and qRT‐PCR Correlation

Microarray (1‐c) v qRTPCR

0.98

1(r)

Microarray (2‐c) v qRTPCR

0 9

0.92

0.94

0.96

Correlation

FC < 1.3 FC > 1.3 FC>2.6

0.9

FC < 1.3 FC > 1.3 FC>2.6

Microarray (1-color) versus qRTPCR

0.9613(20 entities)

0.9585(9 entities)

0.9782(6 entities)

Microarray (2-color)versus qRTPCR

0.9601(18 entities)

0.9702(11 entities)

0.9759(9 entities)


versus qRTPCR (18 entities) (11 entities) (9 entities)

Agilent Gene Expression Provides High Reproducibility• Agilent arrays provide consistent results from technical Agilent arrays provide consistent results from technical

replicates, for data you can trust

Agilent 4x44K Nimblegen 12x135K

HEK293 MAQC A MAQC B

EK29

3

HEK293 MAQC A MAQC B

HEK

293

HE

MAQC A

MAQC A

MAQC B

MAQC B


SurePrint G3 8x60K vs SurePrint HD 4x44K Microarray

• The new SurePrint G3 microarrays provide 1-color data• The new SurePrint G3 microarrays provide similar signals and log ratios as compared to existing 4x44K microarrays

– For sensitive and reliable expression detectionm = 1.037R = 0.985g

1 color dataLog2 (B/A)

For sensitive and reliable expression detection

• SurePrint G3 arrays enable

– Higher density, more content

N = 20468

x6

0K

, 2

5 n

Higher density, more content

– Lower RNA input

m = 1 003

8x

m = 1 028

2-color dataLog2 (B/A)

m = 1.003R = 0.981N = 12729

K,

10

ng

m = 1.028R = 0.985N = 12759

K,

10

ng

8x6

0K

8x6

0K

4 44K 2 25 4x44K v2, 25 ng4x44K v2, 25 ng


LIQA Comes in Both One-Color and Two-Color Options

P/N Product Name Description Dyes

5190‐2305 Low Input Quick Amp Labeling Kit, one‐color Cy35190 2305 Low Input Quick Amp Labeling Kit, one color Cy3

5190‐2306 Low Input Quick Amp Labeling Kit, two‐color Cy3 + Cy5

Kits are for 24 reactions per color

• 1-color kits include 1 enzyme module + Cy31 color kits include 1 enzyme module + Cy3

• 2-color kits include 2 enzyme modules + Cy3 + Cy5

This means:

• 24 arrays for 1 color (24 reactions total)• 24 arrays for 1-color (24 reactions total)

• 24 arrays for 2-color (48 reactions total)


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