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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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)
SurePrint HD 4x44K v2 34,017 Ø 10 Agilent
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)
SurePrint HD 4x44K v2 26,930 Ø 10 Agilent
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
For research use only. Not for use in diagnostic procedures
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)
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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 $$ $$$
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
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f Genes D
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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)
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
) 25000
30000
35000
40000
Num
berNum
ber25762
34617
33686
36790
3567134912
30086
2817726130
2701126061 27096
28177
25
30
35
40
) 25000
30000
35000
40000
Num
berNum
ber25762 2613025466 25116
15
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)
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25000
r of Genes D
eter of G
enes Dete
25762 2613025466 25116
15
20
25
CV (%
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15000
20000
25000
r of Genes D
eter of G
enes Dete
0
5
10
07 16 RR ES RA 07 16 RR ES RA 07 16 RR ES RA
0
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ectedected
0
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10
07 16 RR ES RA 07 16 RR ES RA 07 16 RR ES RA
0
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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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
*
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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)]
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
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40N
umb
Low Input Quick AmpQuick Amp
3151230977
3332531892
34051
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ber of Detec
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0C cted Genes
0
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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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
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d Si
gnal
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%C
V Pr
oces
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oces
sed
251503410073 251503410087
1 2 3 4
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01 2
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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
For research use only. Not for use in diagnostic procedures
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)
For research use only. Not for use in diagnostic procedures
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)
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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
For research use only. Not for use in diagnostic procedures
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)
For research use only. Not for use in diagnostic procedures
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