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Arcturus LCM Instruments and Microgenomics Reagents
Arcturus® LCM Instruments and Microgenomics Reagents Steven Blakely, Product Manager May 6, 2009 Arcturus® LCM Instruments (2006) 1997 (2007) 2009 AXT Live Cell Module (2009) PixCell (1997) PixCell II (1998) PixCell IIe (2000) Veritas (2004) AutoPix (2002) ArcturusXT (2007) ArcturusXT-Ti (2008) AutoScanXT Image Analysis (2007) AXT Motorization, AXT and Veritas Stage Inserts (2008) ArcturusXT™ Microdissection Instrument Flexible and Modular Dual IR and UV LCM System ArcturusXT™ Microdissection Instrument MDS exclusive IR laser-enabled LCM and UV laser cutting Nikon® Eclipse Ti-E inverted research microscope base Simple user interfaces Phase contrast and Differential Interference Contrast (DIC) Superior optics: 2X – 100X (air and oil) Binoculars for standard microscope functions Optional high resolution imaging camera, including MetaVue Imaging System AutoScanXT Image Analysis Software Module Open, modular design – each system tailored to user needs Validated System for Microgenomics® with full applications support Phase Contrast Images courtesy of Nikon DIC Arcturus LCM Instruments – The Best of Both Worlds Arcturus LCM systems are the only commercially available platforms to offer both IR and UV lasers IR Laser Capture (LCM) Exclusive to MDS Analytical Technologies Delivers a gentle technique, ensuring bio-molecule integrity Best choice for single cell or small number of cells Allows reliable use of plain glass slide preparations Never lose custody of your sample! UV Laser Cutting Before LCM After LCM Provides additional speed and flexibility Ideal for non-soft tissues and large number of cells Allows use of membrane slide preparations Contact or non-contact microdissection CapSure Cap ArcturusXT Neurological Slide Stage Insert Optimized for Neurobiology Large format slide stage insert Common neurological slide preparations User-adjustable positions 25 mm, 38 mm and 50 mm NEW! ArcturusXT Live Cell Application Module NEW! NEW! ArcturusXT Live Cell Imaging Nikon Eclipse Ti-E research microscope base Optional high resolution camera with MetaMorph imaging software Fluorescence imaging Contrast imaging Phase contrast or DIC Phase Contrast DIC ArcturusXT Live Cell Microdissection NEW! NEW! ArcturusXT Live Cell Microdissection REPLACE 0 Hours Post-Microdissection RNA Yield: 2.7 ng/μl RIN: 9.5 24 Hours Post-Microdissection RNA Yield: 2.6 ng/μl RIN: 9.6 48 Hours Post-Microdissection RNA Yield: 3.1 ng/μl RIN: 9.7 Cultured CHO cells Æ Microdissection does not affect cell or nucleic acid viability Arcturus LCM Instruments and Microgenomics Reagents Fueling Discovery in Diverse Research and Therapeutic Areas • Areas of research • • • • • • • • • • • • Oncology Developmental Biology Neuroscience Proteomics Forensics Inflammation Cardiovascular Diabetes Live Cells Hard tissues (bone, skin, cartilage) Sub-cellular structures (chromosomes, organelles, etc) Plant Arcturus LCM Applications Gene Expression Microarray Mutation Analysis Real-Time PCR Gene Expression Analysis Proteomic 2D Gels Ciphergen Proteomics – Protein Chip/Mass Spec Challenges of Small Sample Research Collect a pure cell population from heterogeneous tissue LCM Obtain enough material for analysis Minimize sample loss during processing Microgenomics Maintain biomolecule integrity Reproducible analysis Quality control What is Microgenomics? Microgenomics Quantitative genomic (and proteomic) molecular analysis of single cells or small groups of cells Systems for Microgenomics® Integrated and complete sets of instruments, reagents and protocols for the study of microgenomics Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS AT Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, Paradise PLUS Amplification RiboAmp® PLUS family, Paradise PLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. HistoGene® LCM Frozen Section Staining Kit HIGH QUALITY mRNA AND PROTEIN FROM FROZEN SECTIONS Mouse Intestine Mouse Kidney HistoGene® LCM Immunofluorescence Kit RNA PRESERVATION & EXCELLENT VISUALIZATION Dehydration only Human jejunum + anti-cytokeratin 2.5 F luorescence 2.0 Conventional Immuno-staining 1.5 1.0 0.0 19 24 29 34 28S 18S 0.5 39 Time (seconds) 44 49 54 59 0.40 0.35 0.30 0.25 0.20 0.15 0.10 HistoGene Immunofluorescence Kit 0.05 0.00 19 24 29 34 39 Time (seconds) 44 49 54 59 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.00 19 24 29 34 39 Time (seconds) 28S 0.25 18S Mouse small intestine + anti-MAdCAM-1 F lu o r e s c e n c e F lu ore sce nce Inflamed mouse lacrimal gland + anti-CD4 44 49 54 59 Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, ParadisePLUS Amplification RiboAmp® PLUS family, ParadisePLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, ParadisePLUS Amplification RiboAmp® PLUS family, ParadisePLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. PicoPure® DNA Isolation Kit OPTIMIZED FOR SMALL SAMPLE RECOVERY No purification needed prior to PCR Cycle thresholds for the PicoPure Kit samples were 28.9 ± 0.28 and 32.4 ± 1.35 for the column-purified samples, respectively. Samples amplify earlier (more DNA present) Samples amplify more consistently (less sample variation) PicoPure RNA Isolation Kit OPTIMIZED FOR SMALL SAMPLE RECOVERY 100μg capacity Total RNA from 1 cell Replicate samples of 1, 10, 100, and 1000 cells were microdissected using the PixCell IIe LCM Instrument. RNA was isolated using the PicoPure RNA Isolation Kit, reverse-transcribed and subjected to a QRT-PCR assay for GAPDH using a LightCycler (Roche). Quantifiable message can be detected from a single cell, and the fluorescence signal is proportional to the cell number for all samples studied. Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS AT Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, ParadisePLUS Amplification RiboAmp® PLUS family, ParadisePLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. RiboAmp®Plus RNA Amplification Kits Amplify enough RNA for microarray analysis consistently and robustly from a few nanograms of sample RiboAmp Plus Î 5 ng - 40 ng RiboAmp HS Plus Î 100 pg - 5 ng aRNA yields enough material for a microarray (up to 50 μg) with 2 rounds of amplification RiboAmp is now available in a wide variety of configurations customized for your research: Æ Biotin labeling Æ Cy3 Labeling Æ Cy5 Labeling Æ Amino-allyl incorporation Æ Natural nucleotides Æ qrtPCR Paradise®Plus Reagent System • The Challenge: Analysis of Archived Material • Vast majority of biopsies are fixed in 10% formalin and paraffin-embedded (FFPE) Formalin-fixation Cross-linking by aldehyde groups affects structural integrity of nucleic acids and proteins Best for working with DNA Presents additional challenges when working with RNA Paraffin processing and embedding Extraction of nucleic acids from paraffin embedding causes degradation of RNA and DNA • Until recently, no method existed for gene expression array analysis of FFPE samples • The Solution: Arcturus’ Paradise Plus FFPE Reagent System • Gene expression studies of FFPE samples are possible • Retrospective and prospective studies • The Paradise Plus system is analogous to: • HistoGene Staining, • PicoPure RNA isolation and • RiboAmp PLUS Amplification … In one kit optimized for FFPE material Paradise®Plus Product Family • Paradise® Plus Reagent System – Gene Expression Analysis for FFPE Specimens and LCM Samples • Paradise® Plus QC Kit – Qualify your FFPE samples • Paradise® Plus Quantitative Real Time PCR (qRT-PCR) Kit – Quantify Your Genes of Interest • Paradise® Plus Whole Transcript Reverse Transcription (WT-RT) Reagent System – Formulated for Real-Time PCR (RT-PCR) Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS AT Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, ParadisePLUS Amplification RiboAmp® PLUS family, ParadisePLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. Turbo Labeling™ Kit Non-enzymatic technology for labeling of DNA or RNA for any microarray application including Oligo, cDNA and CGH arrays Easy protocol takes less than an hour Allows splitting samples for comparative studies and differential labeling Especially useful when sample needs to be amplified Enables using natural nucleotides in the amplification process Results in unmodified aRNA with higher yields and longer aRNA fragments Better representation of the mRNA transcript for downstream analysis Higher %P calls Turbo: How it labels Technology is based on the stable binding properties of a platinum complex to biomolecules. Label consists of a platinum complex, a detectable molecule and a leaving group that is displaced upon reaction with the target This molecule forms a coordinate bond, firmly coupling the label to the target The label is bound to DNA or RNA by binding to the N7 position of guanine Systems for Microgenomics® Microgenomics Process Sample Collection Specimen Preservation MDS AT Products Biopsy, FACS sorting, cell culture EtOH/Frozen, FFPE Cell Identification HistoGene®, Standard and IF, Paradise® PLUS Cell Selection PixCell®, AutoPix®, Veritas™, ArcturusXT™ Extraction and Purification PicoPure® RNA, DNA, Paradise PLUS Amplification RiboAmp® PLUS family, Paradise PLUS Labeling Molecular Analysis Turbo™ Biotin, Cy™3, Cy™5 Microarray, PCR, QPCR, Tissue Array, 2-DGE, LC/MS, etc. Complete Solution for Gene Expression Analysis Cell Identification Cell Selection Cell Isolation and Extraction HistoGene ArcturusXT PicoPure Amplification RiboAmpPLUS ParadisePLUS Labeling Turbo Analysis GenePix Q&A
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