Sensitive Gene Expression Using Minimal RNA Amounts Gene Expression Using Minimal RNA Amounts ......

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  • Sensitive Gene Expression Sensitive Gene Expression Using Minimal RNA Amounts

    Introducing Agilents 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

    Agilents 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 m features

    Compatible with:Agilent B or C Scanner

    30 m 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 Agilent96 ERCC

    Refseq Build 36.3;

    Human8x60K , , 96 ERCC

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

    SurePrint HD 4x44K v2 27,958

    10 Agilent12 ERCC

    SurePrint G3 10 Agilent

    Mouse

    SurePrint G3 8x60K 34,017 4,623

    10 Agilent96 ERCC

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

    SurePrint HD 4x44K v2 34,017

    10 Agilent12 ERCC

    Rat

    SurePrint G3 8x60K 26,930

    10 Agilent96 ERCC

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

    SurePrint HD 4x44K v2 26,930

    10 Agilent12 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

  • 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

    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

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    atio

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