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Gene Expression and DNA Copy Number Analysis in Plants Gene Expression and DNA Copy Number Analysis in Plants QuantiGene® Plex Assay Use for quantitative analysis of both multiplexed gene expression and DNA copy number variation. Sample types Applications Plant tissues, root, stem branch, leaves, seeds Plant breeding Protoplasts Identification of transgenic, knockout, siRNA Cultured cells Zygosity testing Isolated DNA/RNA GMO testing Many others Viral, fungal, and bacterial testing QuantiGene Plex Assays combine branched DNA signal amplification technology and xMAP® (multi-analyte profiling) beads from Luminex® to enable simultaneous direct quantification of multiple RNA or DNA targets from a variety of sample types. bDNA technology is a sandwich nucleic acid hybridization assay that provides a unique approach to RNA and DNA detection and quantification by amplifying the reporter signal rather than the template By measuring the RNA or DNA at the sample source, the assay avoids biases and variability inherent to extraction and amplification of target sequences. How QuantiGene® Plex Assays work Sample preparation Target hybridization Luminex® beads with Capture Probes Signal amplification PreAmplifier Amplifier Incubate beads with sample and probes Detection Streptavidin phycoerythrin (SAPE) Read signal using a Luminex® instrument Label Probe 80 RNA or DNA targets per well Capture Extenders (CE) Lyse sample and go Hybridizations and wash steps Label Extenders (LE) Blocking Probes (BL) Lysate (with target RNA or DNA) Lysate (with target RNA or DNA) Features and benefits Any gene, any species – Customers have utilized our QuantiGene Assays to measure gene expression and DNA copy variation from wheat, corn, soybean, rice, tobacco, lettuce, potato, tomato, cherry tomato, Arabidopsis, and many others Fast customization – if we do not have the gene(s) in stock, we can create your custom panel within 2 weeks True multiplexing – Measure up to 80 genes of interest and housekeeping genes in the same well with no crossreactivity 96- or 384-well plate format with results in 1.5 days No PCR – No RNA and DNA purification, no reverse transcription, no PCR amplification, and none of the associated artifacts Standardized platform – 96-well plate format compatible with Luminex 100, 200, MagPix® and FlexMap3D® systems Simple workflow – ELISA-like workflow for direct hybridization of transcripts to beads and transcript labeling 2 Gene Expression - Soybean Expression of AtBBX32 in soybean affects the transcript abundance of central clock components near ZT 0. Levels of both central clock components GmLCL2 (A) and GmTOC1 (B) were assayed by quantigene RNA extraction and expression analysis from V2 leaf tissue harvested from soybean plants grown in a controlled environment. Growth chamber experiment was performed in a 14:10 hour photoperiod (Light:Dark) with 650 mE of light. p-values based on the difference between both transgenic lines and wildtype control. * p#0.05. Where error bars are not visible they are smaller than the data points. Reprinted from Preuss SB, et al., Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield, PLoS ONE 7(2): e30717 (2012). doi:10.1371/journal. pone.0030717.g003 DNA Copy Number - Lettuce Results of the QuantiGene Plex DNA Assay with different sample sources, including purified genomic DNA (gDNA) as well as lysates from seeds (seed) and leaves (leaf) of a transgenic lettuce plant. Normalized data with gene 10 are shown to indicate the relative DNA ratio. 1.2 Normalized to gene 10 1 0.8 Mar gDNA 300 ng 0.6 Mar Seed 40 μl 0.4 Mar Leaf 40 μl 0.2 0 Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Gene 7 Gene 8 Gene 9 Gene 10 3 Specifications Gene expression Limit of Detection ≤1,000-2,000 transcripts/assay well Limit of Quantitation ≤2,000-4,000 transcripts/assay well Linear Dynamic Range ≥3.0 logs Assay CV ≤15% intra-assay; ≤20% inter-assay Compatible Sample Types Cultured cells, plant tissue, protoplasts, purified RNA Assay Format 96- or 384-well plate Targets/well 3- to 80-plex for both gene expression DNA Copy Number Limit of Detection ≤10,000 copies/assay well Limit of Quantitation ≤20,000 copies/assay well Linear Dynamic Range ≥2.5 logs Assay CV ≤ 5% intra-assay; ≤20% inter-assay Compatible Sample Types Cultured cells, plant tissue, protoplasts purified DNA Assay Format 96 or 384 well plate Targets/well 3-80 genes References Preuss, S.B., et al. Expression of the Arabidopsis thaliana BBX32 Gene in Soybean Increases Grain Yield, PLoS ONE 17 Feb 2012, 7(2): e30717 Armstrong, T.A., et al. Quantification of Transgene-Derived Double-Stranded RNA in Plants Using the QuantiGene Nucleic Acid Detection Platform, J. Agric. Food Chem., 2 Dec 2013, 61(51) : 12557–12564 Cui, C., et al. Determination of Target Gene Copy Number in Plants Using QuantiGene Branched DNA Technology, ASA-CSSA-SSSA International Annual Meeting, Nov, 14, 2006, #162-1 Custom panels Custom panel sets are available for any gene and any species. Custom panels ranging from 3- to 80 plex can be designed and shipped within 2 weeks. Customers simply provide Affymetrix with a gene list, RefSeq IDs, or DNA sequence. eBioscience (US) Tel: +1-888-999-1371 Affymetrix, Inc. Tel: +1-888-362-2447 Tel: +1-858-642-2058 eBioscience (EU) Tel: +43 1 796 40 40 305 Affymetrix UK Ltd. Tel: +44-(0)1628-552550 Panomics Solutions Tel: +1-877-726-6642 panomics.affymetrix.com [email protected] Affymetrix Japan K.K. Tel: +81-(0)3-6430-4020 USB Products Tel: +1-800-321-9322 usb.affymetrix.com www.ebioscience.com Please visit our website for international distributor contact information. For Research Use Only. Not for use in diagnostic or therapeutic procedures. QG03548-1- PLF QGPPX Plant 0414 ©Affymetrix, Inc. All rights reserved. Affymetrix®, Axiom®, Command Console®, CytoScan®, DMET ™, GeneAtlas®, GeneChip®, GeneChip-compatible™, GeneTitan®, Genotyping Console™, myDesign™, NetAffx®, OncoScan™, Powered by Affymetrix™, PrimeView®, Procarta® and QuantiGene® are trademarks or registered trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners. BestProtocols®, eBioscience®, eFluor®, Full Spectrum Cell Analysis®, InstantOne ELISA™, OneComp eBeads™, ProcartaPlex™, Ready-SET-Go!®, SAFE™ Super AquaBlue®, The New Standard of Excellence® and UltraComp eBeads™ are trademarks or registered trademarks of eBioscience, Inc. Instant ELISA® is a registered trademark of Bender MedSystems, GmbH. FlowLogic® is a registered trademark of Inivai Technologies. Luminex®, xMap®, and MagPix® are all registered trademarks of the Luminex Corporation. Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334; 6,140,044; 6,399,365; 6,420,169; 6,551,817; 6,733,977; 7,629,164; 7,790,389 and D430,024 and other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637 and 6,054,270. Cyanine (Cy) dye conjugates are covered by US Patent Nos. US5,569,587 and US5,627,027.