LCM Protocols and Techniques LCM User Meeting Shirley Chu Applications Scientist Arcturus LCM Products

Transcription

1 LCM Protocols and Techniques 2009 LCM User Meeting Shirley Chu Applications Scientist Arcturus LCM Products

2 Agenda Tissue Preparation Tissue Sectioning Staining Specialized Applications

3 The Challenges of Tissue Prep for LCM Fluorescence Got This But Want This? Fluorescence Time (seconds) Time (seconds)

4 Gene Expression Analysis: Frozen v. FFPE #62-FFPE Agilent Microarrays #62-Frozen Expressed genes = 6790 R = #27-Frozen #27-FFPE Expressed genes = 3477 R = Coudry et al., JMD 2007

5 Preparing Samples for Laser Capture Microdissection Many common histological preparations are compatible with laser cutting and laser capture microdissection Downstream analysis of microdissected samples include RNA, DNA and protein For microgenomic analysis, the sample preparation process must facilitate cell identification and preserve the integrity of biomolecules of interest

6 General Guidelines for Specimen Preparation Tissue should be processed as soon as possible upon removal All cells contain proteases including nucleases that are still active after removal or upon death Autodegradation of biomolecules RNase-Free conditions should be observed at all times during handling of tissues and sections.

7 RNase-Free Technique Wear disposable gloves and change frequently Use new or clean instruments between each animal or patient specimen Use RNase-free or Nuclease free solutions, glassware and plasticware Use RNase AWAY or similar product to clean equipment

8 Frozen Tissue Preparation Tissues should be frozen in OCT or a similar product Preferred Freezing Methods Isopentane cooled over liquid Nitrogen Isopentane cooled with dry ice Other Freezing Methods Dry Ice Alone (not optimal, slow) Liquid Nitrogen (not optimal, sectioning difficulties) Cryostat (NO!!!) Tissues can be sectioned immediately or stored in a 70C freezer

9 Frozen Tissue Sectioning Gloves must be worn at all times Cryostat must be cleaned prior to use. All surfaces must be wiped down with % ethanol, especially knife holder and anti-roll plate Recommended Section thickness LCM alone = 8-10um LC+LCM = up to 200um Mount sections onto room temperature slides. After mounting the sections place in slide box stored on dry ice. SLIDES MUST REMAIN COLD!!! For mounting of sections onto frame membrane slides refer to Arcturus Protocol #9 Use separate areas of the microtome blade for each specimen Sections can be stored in a 70C freezer until further processing

10 Formalin-Fixed Paraffin Embedded (FFPE) Tissues 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

11 FFPE Tissue Processing Fixation in 10% Neutral Buffered Formalin as soon as possible after harvesting Fixation should not exceed 24 hours at room temperature with tissue thickness not exceeding 5mm during fixation process Tissues should be processed into paraffin immediately after fixation. Storage in ethanol or PBS is not recommended

12 Preparation of Tissue Sections General Guidelines Clean all equipment and instruments with RNase Away or similar product. Exception: Cryostat should be wiped down with % Ethanol Use separate area of microtome blade for each sample to prevent cross contamination Discard first 3-4 sections from tissue block Section thickness: LCM only 5-10µm; UV Laser Cutting up to 100µm Frozen Tissues Mount sections directly onto room temperature slides. For frame membrane slides, refer to Application Note #9 Do not allow slides to come to room temperature prior to initiating staining process. Hold slides in slide box on dry ice or within cryostat Store slides at 70C Formalin Fixed Paraffin Embedded Tissue Use Nuclease Free or DEPC treated water for tissue floatation bath Float sections for minimal amount of time, no more than 1-2 mins Once sections mounted on slides, prop up vertically to allow water to drain away from sections Air dry for about 2 hrs at room temperature. Do not use oven to dry sections. Slides can be store for up to 2wks at room temperature with dessicant, for longer terms store at 70C.

13 Whole Mount Preparations Cell Culture Blood smears Fine Needle Aspirates Chromosome Metaphase Spreads Sample preparation by cytocentrifugation Ethanol or Formalin fixation possible Use immediately after staining for RNA isolation Stained slide can be stored in a dessicator for DNA isolation

14 Optimal Staining vs. RNA Quality Visualization RNA Quality

15 Staining and Dehydration Staining Recommended for LCM Light staining of tissue sections improves visualization Staining agents and protocols can affect quality and yield of recovered molecules Some samples do not require staining ex. GFP label Dehydration Mandatory for LCM Removal of water inactivates nucleases Graded ethanol series 75%, 95%, 100% Ethanol concentrations must be freshly prepared Xylene steps performed in fumehood Air Drying no more than 5 minutes in a fume hood Provides conditions that maximizes the LCM process

16 General Staining Guidelines (Frozen and FFPE) Total staining time should be as short as possible Staining dishes should be nuclease free Staining solutions should be dedicated for use with LCM samples Solutions are prepared with nuclease free water Stained slides can be held in xylene until initiation of laser capture microdissection Once removed from xylene, microdissection should be completed within 2 hours

17 Histochemical Staining HistoGene Stain for Frozen Tissue 75% ETOH - 30secs NF dh secs Histogene stain secs NF dh secs 75% ETOH - 30 secs 95% ETOH - 30 secs 100% ETOH - 30 secs Xylene - 5 mins Total Time = 8.5 mins Aqueous Time = 3.5 mins Paradise Stain for FFPE Tissue Xylene 2-3 mins Xylene _ 2-3 mins 100% ETOH 1 min 95% ETOH 1min 75% ETOH 1 min NF dh secs Paradise Stain secs NF dh secs 75% ETOH 30 secs 95% ETOH 30 secs 100% ETOH 1 min Xylene 5 mins Total Time = 18 mins Aqueous Time = 7 mins

18 HistoGene Immunofluorescence Kit 1 o Antibody tissue antigen Cy3 streptavidin Total staining time = 17 mins biotin

19 Stains Compatible with Downstream RNA Analysis HistoGene Frozen Section Staining Kit Paradise Kit for FFPE Hematoxylin (Mayer s) and Eosin (frozen, FFPE) Cresyl Violet (frozen sections) Toluidine Blue (frozen section) HistoGene Immunofluorescence Staining Kit

20 RNA Quality Assessment of Tissues Frozen Tissues: Arcturus Protocol #1-Tissue Scrape Protocol for Verifying RNA Quality FFPE Tissues: Paradise Plus or Paradise Plus WT-RT Kits Take a look at the RNA in your tissue sample prior to LCM and downstream analysis Analysis can be completed from as little as one tissue section RNA NanoDrop: Quantity Agilent Bioanalyzer: Quality qrt-pcr: 3 /5 ß-Actin Ratios, transcribe ability of the RNA (important with FFPE samples)

21 Arcturus LCM The Best of Both Worlds Arcturus LCM systems are the only commercially available platforms to offer two 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 UV Laser Cutting 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

22 Slide Formats for LCM and LC/LCM Multiple modes for microdissection. Enables flexibility w/ applications. Removes dependence on tissue dehydration. Standard Glass Glass Membrane Frame Membrane

23 Compatible with ALL standard glass and membrane slides Standard Glass Slides Glass Membrane Slides Frame Membrane Slides LCM & UV Ablation LCM, LCM & UV Ablation LCM & UV Ablation IR Laser LCM IR Laser LCM IR Laser LCM CapSure CapSure CapSure Tissue Glass Slide Membrane Tissue Slide Frame Tissue Membrane Glass Slide UV Laser Cutting and Ablation UV Laser Cutting and Ablation UV Laser Cutting and Ablation

24 Should I Use Capture (LCM) or UV Cutting? Depends on Sample Type Sample Area Number of cells Downstream application

25 IR Laser Capture vs UV Laser Cutting Choice Based on Experimental Objective Large Area Captures IR - Laser Capture UV - Laser Cutting Same size dissections 650μm diameter Comparable RNA quality UV laser cutting enables quick dissection of large areas and hard tissues

26 IR Laser Capture vs UV Laser Cutting Choice Based on Experimental Objective Small Area Captures IR - Laser Capture UV - Laser Cutting Same size dissections 30μm diameter Differences in RNA quality IR Laser Capture preserves RNA quality for small areas and single cell dissections

27 Cell Quantity Recommendations (frozen sections) DNA Analysis (PCR-based) ~250 cells RNA Analysis RT-PCR Low abundance genes - >50,000 cells High abundance gene - ~250 cells Linear Amplification cells (RiboAmp Plus) cells (RiboAmp Plus HS) cells (RiboAmp Plus OA) Protein Analysis 2D gels - >25,000 cells Westerns 10,000 cells Mass spec - 5,000 cells

28 Cell Quantities and Downstream Applications Source: Espina V etal, Laser-Capture Microdissection, Nature Protocols (1:2),

29 IR Laser Capture Microdissection of Single cells Before LCM Frozen Mouse Brain Histogene Stain After LCM CapSure Cap

30 Veritas UV Laser Cutting - large area dissection Before Laser Cutting FFPE Human Breast Tissue After Laser Cutting CapSure Cap

31 Arcturus XT Laser Cutting Live Plant Microdissection Whole Mount of a Blade of grass on frame membrane slide.

32 UV Laser Cutting of Subcellular Organelles Before Laser Cutting Human Chromosome Metaphase Spread Giemsa stained After Laser Cutting CapSure Cap

33 LCM of GFP Expressing Embryos Application Note #3 LCM of Cells Labeled with Enhanced Green Fluoresence Protein Courtesy of: Vasker Bhattacherjee etal, University of Louisville

34 Double ImmunoFluorescence (IF) Images courtesy of: Casey Vickers, PhD Candidate, Baylor College of Medicine

35 Live Cell Microdissection Arcturus XT Arcturus Application Note #11 Isolate living cells for reculture or molecular analysis 3T3 cells visualized with DIC optics

36 Thank You for Attending! For Technical Support please contact us: Toll-free (US): Direct (International): For additional LCM Protocols and Applications Literature Citation Database is searchable and has citations for all LCM related publications