The Veritas Laser Microdissection System

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The Veritas Laser Microdissection System
Veritas Training and Sample Preparation
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We Thank You For Your Business!
The MDC LCM Team
Technical Support
Sales Inquiries
Miles Bellamy or Kristen Schmidt Technical
Applications Scientists [phone]
x345/337 Miles.Bellamy_at_moldev.com
Joseph Pham, Inside Sales Account Manager (888)
446-7911 x391 Joseph.Pham_at_moldev.com
Applications Training
Account Representative
James Grabau,
Deren Lomago, Field Applications
Specialist C(724) 466-0112 Deren.Lomago_at_moldev.com
Midwest Sales Representative
[phone]
James.Grabau_at_moldev.com
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Agenda

• Why Microdissect?
• Arcturus Technology (from NIH)
• The Instrument and How to Use It
• Tissue Preparation
• Extraction Techniques
• Linear Amplification

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Why Use Laser Capture Microdissection (LCM) in
Gene Expression Studies?
To reveal accurate, cell-specific expression
profiles otherwise obscured in mixed cell samples
Pure populations vs. Mixed populations
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The LCM Process

• Visualize Biopsies

• Identify Target Cells

• Isolate Target Cells
• by LCM

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LCM Allows Tumor Specific Gene Expression
Analysis
Normal Breast Tissue
Breast Carcinoma
LCM Uncovers Breast Cancer-specific Molecular
Signatures
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The Power of Microgenomics in Cell-Specific
Molecular Analysis
Homogeneous cell populations reveal hidden
molecular signatures
Gene 1 Proteoglycan 1 Gene 2 CD53
antigen Gene 3 Proteosome Subunit
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Advantages of LCM

• Low energy near-IR laser does not damage proteins
  or nucleic acids.
• Single cells, small of cells and intricately
  shaped areas are easily captured.
• Maintains the morphology of captured cells.
• Adjacent tissue is neither altered or destroyed.
• Contact capture method cells are precisely
  located, inspected and imaged after
  microdissection.

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The Big Picture

• 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

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Introduction to LCM

• History
• Originated at NIH Invented and patented in 1995
• Primary Inventors Lance Liotta, Robert Bonner,
  Mike E. Buck
• Emmert-Buck, et al., Science 274998-1001, 1996
• Bonner, et al., Science 2781481-3, 1997
• Collaboration between the bioengineering and
  cancer pathology groups of NIH.
• Arcturus was founded in 1996
• NIH LCM Patent was licensed to Arcturus in 1996
• Arcturus only company to license LCM technology

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Arcturus/MDC Milestones

• Founded in 1996

NIH CRADA, PixCell 1997
PixCell II LCM System 1998
Applied Genomics 2000
Microgenomics Systems 2001
AutoPix LCM System 2002
Paradise Reagent System 2003
Veritas LC/LCM System 2004
Joined Molecular Devices 2006

• World-Leading Supplier of Laser Microdissection
  Systems
• Over 1,000 systems installed world-wide
• Approximately 650 peer-reviewed articles

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MDC Microgenomics Applications
Gene Expression Microarray
Mutation Analysis
Proteomic 2D Gels
Real-Time PCR Gene Expression Analysis
Proteomics Protein Chip/Mass Spec
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MDC Integrated Microgenomics System
Staining
Amplification
Purification
Microdissection
FFPE Archives

• HistoGene
• Frozen Tissue
• IHC

• RiboAmp
• High Sensitivity Kit
• Oligo Array Kit
• Standard Kit

• PicoPure
• RNA Extraction/Isolation
• DNA Extraction

• Instruments
• VeritasTM
• PixCell

• ParadiseTM
• Staining
• Purification
• Amplification

Microarrays
PCR
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The Microgenomics Process
MDC Products
Biopsy, FNA, Cytology
FFPE, EtOH/Frozen
HistoGene Products
PixCell, AutoPix, Veritas
PicoPure DNA, RNA and Paradise
RiboAmp Family and Paradise
PCR, QPCR, Microarray, LC/MS
Molecular Signatures
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All Protocols on the Arcturus/MDC Web
Sitewww.moldev.com
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The Veritas TM Microdissection Instrument
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Veritas TM The Latest Technology Platform
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Veritas TM Two Laser System

• Diode IR laser capture (LCM) UV laser cutting
  capabilities for unmatched flexibility,
  precision, and speed
• IR Laser Capture (LCM)
• delivers a gentle technique, maximizing
  bio-molecule integrity.
• Ideal for single cell or small number of cell
  gene expression studies
• UV Laser Cutting
• provides unprecedented speed and flexibility.
• Ideal for non-soft tissues and large of cell
  harvesting for proteomics applications.
• 20X faster than alternative technologies

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How does LCM work?

• Our thermoplastic polymer transfer film
• Absorbs near-IR energy
• Distends predictably, evenly, reproducibly to
  enable selective targeting
• Prevents laser energy from reaching sample
• Becomes adhesive
• Adhesion overcomes opposing forces to enable
  selective capture
• Selective Scotch Tape

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Laser Capture Microdissection Forces
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Laser Capture Microdissection
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Laser Capture Microdissection (LCM)
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LCM Caps

• CapSure Macro LCM Caps
• MacroCap ideal for large area capture
• Contacts tissue surface for optimal adhesion
• 50 microliters of extraction volume needed
• Couples directly with 0.5 microfuge tubes
• CapSure HS LCM Caps
• HS stands for High Specificity
• Optimal for smallers of cells, especially rare
  single cells
• Only 10 microliters of extraction volume needed
• Captures cells with speed and high specificity.
• Use of ExtracSure Device, Alignment Tray,
  Incubation Block

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SEM Image of LCM pulses
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SEM Image of Single Cell Capture by LCM
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SEM Images of LCM Captured Cells
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The Laser Capture Microdissection Process
2. Locate cells
3. Place cap
1. Prepare tissue
4. Pulse laser
5. Remove cap
6. Extract molecules
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The Extraction of BioMolecules
Pipette Lysis Buffer
Incubate Sample
Pipette tip
0.5 ml Micro- centrifuge tube
ExtracSureTM Device
CapSureTM HS LCM Cap
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Veritas UV Laser Cutting
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Single Cell Capture - Veritas IR Laser Capture
Microdissection
Before LCM
Frozen Mouse Brain Histogene Stain
After LCM
CapSure
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Hard Tissue Dissection - Veritas UV Laser Cutting
Before Laser Cutting
FFPE Cresyl Violet - Stained Human Bone Marrow
After Laser Cutting
CapSure
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Plant Tissue Dissection - Veritas UV Laser
Cutting
FFPE X-GlcA Substrate-Stained Arabidopsis Seedling
Before Laser Cutting
After Laser Cutting
CapSure
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Live Cell-Tissue Dissection - Veritas UV Laser
Cutting
400 micron thick Arabidopsis leaf
Before Laser Cutting
After Laser Cutting
CapSure
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Fluorescence Dissection - Veritas UV Laser
Cutting
Before Laser Cutting
Drosophila Embryo X-gal-Stained Fluorescence
After Laser Cutting
CapSure
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Sub-Cellular Organelle - Veritas UV Laser Cutting
Before Laser Cutting
Human Chromosome Giemsa-stained Peripheral
Lymphocyte
After Laser Cutting
CapSure
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Large Area Dissection - Veritas UV Laser Cutting
Before Laser Cutting
FFPE Human Breast Tissue
After Laser Cutting
CapSure
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Laser Cutting vs LCM Choice Based on
Experimental Objective
Large Area Captures
UV Laser Cutting
IR Laser Capture Microdissection

• UV laser cutting enables quick dissection of
  large areas and hard tissues

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Laser Cutting vs LCM Choice Based on
Experimental Objective
Small Area Captures
UV Laser Cutting
IR Laser Capture Microdissection

• IR Laser Capture preserves RNA quality for small
  areas and single cell dissections

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Veritas - Microdissected GFP Neurons
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Double IHC
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Human Chromosome Metaphase Spread (Giemsa
Stained Peripheral Lymphocyte)
Veritas Microdissection System 60X Objective
with High Magnification Upgrade
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Laser Microdissection of Living in vitro Cells

• Optimized on the MDC Veritas Microdissection
  Instrument
• Isolate living cells for reculture or molecular
  analysis
• Cells grown on metal frame PEN membrane slide

• Simple, non-contact protocol
• CapSure Macro LCM Caps

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Laser Microdissection of Living in vitro Cells
Experiment

• Live cells captured using combined IR laser
  capture and UV laser cutting

• RNA was isolated for yield and quality
  assessment
• Average total RNA yield of up to 1 µg
• (2mm diameter area)
• RNA profiles indicate high quality RNA obtained
  from microdissected live cells

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Using The Veritas TM

• The Software
• The Hardware
• Slide Types
• Focusing the IR Laser For LCM
• Spot Size

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• Intuitive Graphical User Interface
• Provides Ease of Use and Performance

1 Load materials with automatic sample
detection 2 - Adjust microscope using autofocus
and automatic light adjustment 3 Draw dissect
manually or using AutoScan
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Key Features of VeritasTM

• Revolutionary Graphical User Interface for Power
  and Ease

Flexible Microdissection Choices with IR laser
capture, UV laser cutting, UV ablation

Automatic Materials Detection - 3 slides , 8
Caps bar code compatibility
Automated Microscope Controls with Autofocus
Autobrightness, Autofluorescence
Real time live static image roadmap to live
image tracking
Integrated Study Tree for image and data
Good Laboratory Practice
Capture Groups with µm2 area calculations for
all regions
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Flexible and Automated Materials Handling
Compatibility with ALL slide types glass
and membrane Easy slide naming with built-in
bar-code integration
Automatically detects caps and slides to
ensure accuracy Integrated QC station for
easy CapSure inspection or ablation of unwanted
material
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Automated Microscope Controls For Fast and Easy
Adjustments
Autolight Intensity produce the best picture
every time! Automatic fluorescence filter
wheel integration

• Autofocus - removes the guess work!
• Automatic objective selection with optional 60X
• Stored positions allow for easy tracking of
  dissected areas

Dynamic camera controls with real time color
balancing for the best image quality on
compromised samples
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Flexible Microdissection Tools

• Operate in multiple modes
• - LCM single cells
• - Cut Capture for large areas and hard
  tissues
• - Ablation to remove unwanted material on
  slide OR cap
• Draw regions, freehand or circles with ease

Ruler option to measure in microns Quick
go capture Autoscan integrated automatic
target cell identification and capture
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Capture Groups Record Detailed Statistics of
Harvested Material
Select up to 8 Capture Groups
User selected name, number and total area of
regions in each group
Capture Groups properties
Area of last drawn or selected region
Step thru capture regions
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Materials Loading Detection
Automated Materials Sensors
Modular Insert configured for 3 slides
Load up to 8 caps
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Veritas Modular Sample Tray

• Modular format for 3 standard slides, large
  format slides and possible culture / microtiter
  plate options
• Enclosed inside Veritas to prevent contamination
  of precious samples

Positions for 3 slides
Removable unload station
QC Station
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3 Slide Types

• Compatible with ALL standard glass, membrane,
  new archived slides
• Direct or non-contact operation modes with
  easy, integrated audit trail
• All slides commercially available from MDC

Standard Glass Slides
Membrane / Glass Slides
Membrane Framed Slides
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Sample Preparation Flexibility

• Compatible with ALL standard glass, membrane,
  new archived slides
• Direct or non-contact operation modes with
  easy, integrated audit trail
• All slides commercially available from MDC

Membrane Slides
Standard Glass Slides
Membrane Framed Slides
IR Laser LCM Tack Points
IR Laser LCM
IR Laser LCM
CapSure
CapSure
CapSure
Tissue
Tissue
Frame slide
Membrane
Tissue
Glass Slide
Slide
Glass Slide
Membrane
UV Ablation
UV Laser Cutting and Ablation
UV Laser Cutting and Ablation
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Microdissect a Variety of Samples

• Isolate cells from any tissue
• Compatible with all types of fixed tissues or
  cytological preparations

• Microdissect any morphological shape
• Microdissect single cells or multi-cellular
  structuresregardless of the
• shape (linear, circular, doughnut, etc)

• Preserve cell and tissue integrity
• Microdissected material and surrounding cells can
  never be damaged

• Utilize a variety of slide preparations
  techniques
• Use standard slide preps or previously archived
  samples.

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Focusing the IR Laser

• Laser Focus Mechanics

Focused beam
Unfocused beams with rings
Unfocused beams
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IR Laser
LCM
CapSure HS Cap
Melted Polymer of Cap
Side View
IR Laser Focal Plane
12um stand-Off Rail
Tissue
Tissue
No Tissue portion on slide
Microscope Slide
Top View
No Surface Contact
Dimple Only
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IR Laser
LCM
CapSure HS Cap
Melted Polymer of Cap
Side View
IR Laser Focal Plane
12um stand-Off Rail
Tissue
Tissue
No Tissue portion on slide
Microscope Slide
Crisp Ring
Top View
Surface Contact
(inner circumference)
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The LCM Spot

• Ensure film is able to contact the glass.
• Take a practice shot where there is no tissue.
• Adjust IR laser power and duration settings to
  insure polymer film touches the glass.
• If film touches glass IR laser and LCM cap are
  ready for for capture.

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Proper Laser Focus and LCM Spots
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LCM Sample Preparation

• Tissue Preparation
• Slide Preparation
• Staining Techniques

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Preparing Samples for Laser Capture

• Many common histological preparations are
  compatible with laser capture microdissection.
• For microgenomic analysis, the sample preparation
  process must facilitate cell identification and
  preserve the integrity of biomolecules of
  interest

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Specimen Preparation

• Tissue should be processed as soon as possible
  upon removal from human or animal.
• All cells contain proteases and nucleases that
  remain active, even after biopsy and cell death.
• RNase-free conditions should be observed at all
  times during handling of tissues and sections.

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RNase-Free Technique

• Wear disposable gloves and change frequently
• 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

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Frozen Tissue Preparation

• Specimen Freezing
• Add a layer of OCT to cryomold.
• Add tissue specimen to OCT.
• Add OCT to cover specimen.
• Freeze the cryomold directly on dry ice.
• Once specimen is completely frozen transfer to
• 80c freezer for storage.
• Snap Freezing in liquid nitrogen
• induces ice crystals
• Alternative Freezing Methods
• Isopentane cooled over liquid Nitrogen
• Isopentane cooled with dry ice
• Petri dish floated on liquid Nitrogen
• Tissue blocks can be sectioned immediately or
  stored in a -80C freezer.

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Frozen Tissue Sectioning

• Take tissue blocks from -80C
• Do all sectioning in a Cryostat
• Use slides that are Room Temperature
• Optimal section thickness is 5-10um for LCM
• Store sections at -80C to preserve RNA or
  proceed w/ LCM
• Do not let sections thaw or air dry prior to
  fixing!!!

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Frozen Tissue Sectioning (cont.)

• Cryostat must be cleaned prior to use. All
  surfaces must be wiped down with 100 ethanol,
  especially knife holder and anti-roll plate.
• Section thickness 5-10 ?m for optimal LCM and
  morphology
• Section thickness 5-400 ?m if doing LCC on
  membrane slides
• Mount on sections on Room Temperature slides.
  After mounting the sections, the slides must
  remain cold
• Use new section of microtome blade for each
  specimen.
• Sections can be stored in a 80C freezer until
  further processing

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Formalin-Fixed Paraffin Embedded Tissue

• Formalin-fixation
• Cross-linking by aldehyde
• groups affects structural integrity
• of nucleic acids and proteins.
• Paraffin processing and embedding
• Extraction of nucleic acids from paraffin
  embedding causes degradation of RNA and DNA.

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Paraffin Tissue Processing

• Fix biopsy in 10 Neutral Buffered Formalin as
  soon as possible after dissection of tissue.
• Fixation should not exceed 24 hours at room
  temperature with tissue thickness not exceeding
  5mm during fixation process.
• Immediately after fixation, tissues must go
  through paraffin embedding procedure.

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Paraffin Tissue Sectioning

• Wipe all surfaces of microtome, waterbath with
  RNase AWAY ? or similar product, followed by 100
  Ethanol.
• Clean out water in tissue floatation bath
  frequently and fill with nuclease free water.
• Float sections for minimal time prior to pick up.
  Dry slides in vertical position, allowing water
  to drain away from sections.

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Paraffin Tissue Sectioning (contd)

• Do not bake or melt sections onto glass slide,
  let sections air dry at room temp.
• Slides can be used after air-drying for 90 min at
  room temperature.
• After sectioning slides store w/ dessicant for up
  to one week at room temperature. For long term
  storage, store at -80C.

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Staining and Dehydration

• Staining Recommended for LCM LCC (Laser Cut
  Capture)
• Light staining improves visualization of dry,
  non-coverslipped slides
• Staining agents and protocols can affect quality
  and yield of recovered molecules
• Some samples do not require staining ex. GFP
  label
• Dehydrating Mandatory for LCM, but not a
  requirement for LCC
• Active 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

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Staining and Dehydration of Both Frozen and
FFPE Samples

• Total staining time should be as minimal as
  possible.
• Staining dishes should be nuclease free.
• Solutions should be 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.

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Staining and Dehydration Frozen Tissue
Colorimetric Stain
Cell Identification
Remove Ethanol
Dehydrate through Graded Ethanols
gt5 min
75 Ethanol
NF Water
NF Water
75 Ethanol
95 Ethanol
Xylene
100 Ethanol
Apply Staining solution
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Staining and DehydrationFFPE Tissue
Colorimetric Stain
Remove Ethanol
Dehydrate through Graded Ethanols
Deparaffinization
NF Water
gt5 min
Rehydrate through Graded Ethanols
NF Water
75 Ethanol
95 Ethanol
Xylene
100 Ethanol
75 Ethanol
Staining
95 Ethanol
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Staining Dehydration Protocol

• Formalin-Fixed Paraffin Embedded sections
• Xylene 5
• Xylene 5
• 100 ETOH 30
• 95 ETOH 30
• 75 ETOH 30
• dH20 30
• Paradise Stain 20-45
• dH20 30
• 75 ETOH 30
• 95 ETOH 30
• 100 ETOH 30
• Xylene 5

• Frozen Sections
• Cell Suspensions
• 75 ETOH 30
• dH20 30
• Histogene stain 20-45
• dH20 30
• 75 ETOH 30
• 95 ETOH 30
• 100 ETOH 30
• Xylene 5

Fix
Deparaffinization
Stain
Dehydration
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Purpose of Dehydration Reagents

• Graded Ethanol solutions
• 75, 95 100 remove water and preserve
  morphology
• Xylene removes ethanol
• Air dry

Use fresh 100 Ethanol for all samples
RNA degrades through RNAse activity and
auto-degradation. This happens fastest when the
sample is hydrated. Dehydration prevents both
types of RNA degradation
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HistoGene? Frozen Section Staining Kit
Staining Techniques

• Mouse Intestine

• Mouse Kidney

• Frozen Tissue Section unprocessed
• HistoGene prepared LCM cells
• Unstained, dehydrated LCM cells
• Agilent BioAnalyzer

• Brilliant high-contract stain optimized to
  preserve RNA integrity

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Requirements to Consider

• Using a stained frozen tissue section
• Must use sample immediately
• Complete LCM session within 2 hours
• RNA quality will degrade rapidly after staining
  has occurred
• DO NOT refreeze a stained tissue section
• Once tissue section has been stained, LCM MUST BE
  PREFORMED
• Partial degradation occurs during any staining
  process.
• Wherever there is an aqueous environment,
• nucleases are active

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IHC Staining Techniques

• HistoGene LCM Immunofluorescence Staining and
  Dehydration Kits

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Challenges

• Standard immunostaining protocols are not
  compatible with retrieval of good quality RNA
• To retrieve good quality and high yields of RNA
  from the stained sample (RNase inhibition, time,
  temperature)
• To achieve staining of good intensity and
  specificity (staining method, antibody and
  conjugate titers)

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RNA Preservation and Excellent Visualization
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10 Minute Immunostaining
antibody
tissue antigen
Cy3
streptavidin
biotin
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Staining and Dehydration - IHC Fluorescent
Staining
Once staining is complete sample cannot be
stored. Use immediately for LCM!!!
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Simplify Fluorescent TargetCell Labeling
Human Prostate Ductal Epithelium prepared with
HistoGene LCM Immunofluorescence Kits
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Alternative Staining Protocols

• Arcturus HistoGene Frozen Section Staining Kit
• Arcturus Paradise Kit for FFPE
• Arcturus Immunofluorescence Staining Kit
• HE
• Cresyl Violet (frozen sections)
• Toluidine Blue (frozen section)

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Extraction Techniques
DNA, RNA, and Protein Extraction and
Isolation

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Challenges of Small Sample Research

• Collect a pure cell population from heterogeneous
  tissue
• Obtain enough material for analysis
• Minimize sample loss during processing
• Maintain macromolecule integrity
• Reproducible analysis

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Extraction and Isolation Techniques w/ CapSure
HS Cap

• The ExtracSure Sample Extraction Device for HS
  Caps

ExtracSure sample extraction is designed to make
efficient use of low volumes of extraction buffer
while sealing out any non-selected material from
the captured cells.
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DNA Extraction From Microscopic Samples

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PicoPureTM DNA Extraction Kit

• DNA Extraction in 4 Easy Steps

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PicoPure DNA Isolation Kit

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DNA Extraction PicoPure DNA

• Extraction Buffer.
• Proteinase K extraction process
• Lyophilized Proteinase K must be freshly prepared
  for each LCM session.
• To be used on any sample type.
• Must go through incubation 65 degrees Celsius.
• Formalin fixed/Paraffin Embedded gt16 hours
• Any other fixation method 3 hours
• After incubation heat inactivate the Proteinase
  K.
• Light Centrifuge.

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RNA Isolation From Microscopic Samples

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RNA Extraction and Isolation

• Extraction Buffer
• Releases RNA into solution
• Disrupts cellular membranes
• Stabilizes RNA
• Denatures proteins including RNases
• Use a GITC beta mercapto-ethanol cocktail.
• Buffers with solvents have found to degrade the
  transfer film off of the LCM cap.
• DO NOT use Trizol. Has been found to degrade the
  LCM transfer film.

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RNA Extraction and Isolation

• Assessing Recovery of RNA
• Completeness of extraction
• Examine cap for unruptured cells
• Examine cap for unextracted nucleic acids using
  fluorescence
• RNA yield and integrity
• Use high-sensitivity, low volume method
  (BioAnalyzer) to assess RNA extractions from
• Pre-protocol tissue scrape
• Large, non-specific population of LCM captured
  cells

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PicoPure RNA Isolation Kit

• Maximize RNA Recovery from Microscopic Samples

• Isolate total RNA from 1 or more LCM cell
• Maintain high-quality RNA
• Elute in 10 µl ready for amplification
• Optimized binding elution chemistry

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PicoPure RNA Isolation Kit

• Components of PicoPure RNA Isolation Kit
• Protocol
• XB Extraction Buffer
• 70 Ethanol
• Wash Buffers 1 and 2
• Elution Buffer
• RNA Purification Columns and Collection Tube
• Microcentrifuge Tube

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RNA Extraction and Isolation

• Features of PicoPure RNA Isolation Kit
• Retain low-abundance mRNA.
• Maintains high quality total RNA.
• Isolate total RNA from 10 or more LCM cells.
• Elute in only 10 µl.
• Sample ready for amplification w/ RiboAmp RNA
  Amplification Kits.
• 2 part kit Extraction and Column Purification.
• Final product is total RNA.
• Final product is ready for RT-PCR or linear
  amplification.

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PicoPure RNA Isolation Kit

• PicoPure RNA Isolation Kit Protocol
• Load onto MiraCol Purification Column
• Spin column to capture RNA
• Wash column twice with wash buffer
• Elute RNA with elution buffer

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PicoPure RNA Isolation Kit

• Troubleshooting RNA Extraction
• Extraction efficiency
• Increase length of 42C incubation
• Do not abbreviate 30 minute incubation
• RNA integrity
• Identify step where loss occurred
• Suspect initial sample quality first.
• Yield and integrity are related
• Degraded RNA has reduced affinity for
  purification column membrane.

102
RNA Quality Check

• Tissue Scrape Protocol
• Tissue scrape represents tissue block. If RNA can
  be found in tissue section,
• then RNA can be found in tissue block.
• Prepare frozen section following protocol for
  LCM.
• Scrape material off of slide extract and purify
  using PicoPure RNA Isolation Kit.
• Run purified product on gel or Agilent
  Bioanalyzer
• Locate ribosomal RNA bands (18s and 28s).
• If bands are found then sample has good RNA.
  Proceed with LCM.
• If bands are weak then sample has degraded RNA.
  Do not LCM.

103

Protein Isolation From Microscopic Samples

104
PicoPureTM Protein Extraction Buffer

• Chaotropic (denaturing) buffer, with high salt
  content.
• Compatible with mass spec and liquid
  chromatography, but use with other applications
  such as 2D gels, western blots, ELISA, etc. would
  require dialysis.
• Product discontinued in 2003.

105
PicoPureTM Protein Extraction Buffer

• 45 mM Tris-HCl
• 0.0188 M EDTA
• 4.61 M GITC
• 1 Triton X-100
• 1 Mercaptoethanol

• Incubate the sample for 3 hours at 65 degrees. 
• After incubation spin down the sample and collect
  the lysate. 

106
Protein Extraction

• Protein and integrity
• Use high-sensitivity, low volume method
  (BioAnalyzer) to assess protein extractions from
• Pre-protocol tissue scrape
• Large, non-specific population of LCM captured
  cells
• Locate protein band
• If bands are found, then sample has good protein
  DO LCM
• If bands are weak, then sample has degraded
  protein NO LCM
• Can also be done by traditional acrylamide gel
  electrophoresis and Western Blot.

107
Amount of Cells Needed

• DNA Analysis (PCR-based)
• 250 cells
• RNA Analysis (from fresh/frozen samples)
• RT-PCR
• Low abundance genes - gt50,000 cells
• High abundance gene - 250 cells
• Linear Amplification
• 500 2000 cells (RiboAmp)
• 50 250 cells (RiboAmp HS)
• 500 3000 cells (RiboAmp OA labeling)
• Protein Analysis
• 2-D gels - gt25,000 cells
• Westerns 10,000 cells
• Mass spec - 5,000 cells

108

Linear RNA Amplification

109
The System for Microgenomics

• Amplify enough RNA for microarray analysis
  consistently and robustly from a few nanograms of
  sample.

Array 1

• 1 to 4 ng total RNA yields 30-50 µg aRNA
• (with 2 rounds of amplification)

Array 2
Array 3
110
RiboAmp Family of Linear RNA Amplification Kits

• RiboAmp RNA Amplification Kit
• RiboAmp HS Amplification Kit
• RiboAmp OA Amplification Kit

111
RiboAmp RNA Amplification Family of Kits
Prepare RNA for Microarrays with Reliability,
Sensitivity and Ease

• RiboAmp OA Kit
• Optimized for oligo array platforms (Affymetrix
  and Agilent)
• Incorporate biotin label or amino allyl label
• RiboAmp HS Kit
• Unparalleled high sensitivity
• Proven for all array platforms

112
RNA Isolation and Amplification
113
RiboAmp Kits - Sample Input
Which Kit Should I Use ?

• RiboAmp ?RiboAmp HS ?RiboAmp OA

114
How Much aRNA Should I Expect?
Assumptions

• 2 of total RNA mRNA
• LCM yields 10pg total RNA/cell
• So
• Start with 100 cells
• 100 cells 1000pg total RNA
• 2 20pg mRNA to start
• 1st Round yields 1-3000x amplification
• One round 20ng aRNA
• 2nd Round yields 1000x amplification
• Two rounds gt20ug aRNA

115
RiboAmp RNA Amplification Process
116
RiboAmp Linear Amplification
Exogenous Primer
1st Round
mRNA
RNADNA Hybrid
T T T T - T7
Primer A
1st Strand cDNA Synthesis
Heat denaturation of original mRNA strand
2nd Strand cDNA Synthesis
In Vitro Transcription (IVT)
117
RiboAmp Linear Amplification
End of 1st Round
2nd Round
RNADNA Hybrid
5
3
3
A A A A
T7 -
T7 - T T T T
5
Exogenous Primer
Antisense Strand
1st Strand cDNA Synthesis
Sense Strand
Heat denaturuation of aRNA strand
2nd Strand cDNA Synthesis
Oligo dTT7 primer
aRNA
1,000 fold Amplification of original mRNA
118
RiboAmp Linear Amplification
2nd Round
T T T T - T7
Double-stranded cDNA
Labeled dNTPs
Commercial Labeling Kit In Vitro Transcription
(IVT)
119
RiboAmp Linear Amplification
End of 2nd Round
Labeled aRNA Target
Ready for fractionization and Hybridization
1,000,000 fold Amplification of original mRNA

• Shorter fragment size than original mRNA
• Antisense orientation
• Biased to the 3 end

120
RiboAmp RNA Amplification Kit
Array 1

• Amplify enough RNA for microarray analysis
  consistently and robustly from a few nanograms of
  sample.

• 1 to 4 ng yields 30-50 µg
• (with 2 rounds of amplification)

Array 2
Array 3
121
Using Formalin Fixed Paraffin-Embedded Tissue

• Tissue Staining
• RNA Extraction and Isolation
• Linear Amplification

The All-In-One System
122
Paradise Reagent System

• The Challenge Analysis of Clinical Biopsies
• Vast majority of biopsies are fixed in 10
  formalin and paraffin-embedded (FFPE)
• No method existed for gene expression array
  analysis of these samples
• The Solution Arcturus FFPE Reagent System
• Gene expression studies of FFPE samples are now
  possible
• Retrospective and prospective studies

123
II. Microdissection
I. Slide Preparation and Staining
Veritas LC/LCM System
III. RNA Extraction and Isolation
V. Microarray and Bioinformatic Analyses
Affymetrix GeneChip probe array. Images
courtesy of Affymetrix.
IV. RNA Amplification
124
Linear Amplification of Matched Frozen and FFPE
Samples

• Amplified aRNA
• Gel Analysis

• Amplified aRNA Yield

Fresh frozen
FFPE

• Provides optimized methods that maximize
  transcribability of cross-linked templates.
• Ability to amplify from 5-10 ng of fixed total
  RNA.

L U 1 2 3 4
125
Robust Amplification Across Sample Types
126
RNA Isolated from Archived Breast Cancer Samples
127
Thank You for Attending!

• For Technical Support feel free to contact us
• Toll-free (US) 888-446-7911
• Direct (International) 650-962-3020
• Additional Protocols/Applications can be found at
  
• www.arctur.com and www.moldev.com
• Literature Citation Database has citations for
  current LCM - related publications.