Arcturus XT Microdissection Systemweb.ecologia.unam.mx/labmicrolas/documentos/6... · 6 | Life Technologies | 11/8/2012 Introduction to LCM Microgenomics Quantitative genomic and

Arcturus® XT Microdissection System

王森右Field Applications Scientist

• Theory of Laser Capture Microdissection

• Introduction to Arcturus XT™ Laser Microdissection System

• Tissue Preparation Seminar

2 | Life Technologies | 11/8/2012

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


2. Identify Target Cells 3. Isolate Target Cells1. Visualize Specimen

LCM of premalignant breast cancer progressionMa, Xiao-Jun et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5974-5979

ADH

Normal

DCIS

IDC

LCM Process


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


Introduction to LCM

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


Microgenomics Process

Biopsy, FACS sorting, cell culture

ArcturusXT™, Veritas™,

PixCell® , AutoPix®

RiboAmp family, Paradise

PCR, QPCR, Microarray, Tissue

Array, 2-DGE, LC/MS, etc.

EtOH/Frozen, FFPE

HistoGene, Standard and IHC

PicoPure RNA, DNA, Paradise

Sample Collection

Molecular Analysis

Cell Selection

Cell Identification

Extraction and Purification

Amplification

Specimen Preservation

Labeling Turbo Biotin, Cy™3, Cy™5


Ciphergen

Arcturus® System LCM Applications Gene Expression –

Microarray Mutation Analysis

Gene Expression –

Real-Time PCR

Proteomics – Protein

Chip/Mass Spec

Proteomics – 2D Gels

Forensics – STR

analysis

Proteomics – Reverse

Phase Protein Arrays


Two fundamental approaches to Microdissection

− IR Laser Capture (LCM)

>Developed at the NIH- exclusive license to Life Tech

>Low energy laser preserves biomolecule integrity (RNA)

>Best choice for single cell or small number of cells

>Allows reliable use of plain glass slide preparations

>Maintains Morphology, nondestructive to adjacent tissue

>Well Published- Over 1,000 citations

− UV Laser Cutting

>Provides additional speed and flexibility

>Ideal for difficult tissues and large samples

>Unwanted material can be ablated

>Supports Contact or Non-Contact Microdissection


How LCM Works

• Near-Infrared (IR) laser activates thermoplastic polymer transfer film

− Thermoplastic film:

> Absorbs IR laser energy

− Prevents laser energy from reaching sample

− Laser energy never directly absorbed by sample

> Becomes adhesive

− Polymer film activates and melts near 70° C

− Sticks to cells of interest

> Increasing IR energy increases activated film area

> Distends predictably, evenly, reproducibly to enable selective

targeting

> Adhesion overcomes opposing forces to enable selective capture

• Cell(s) removed with polymer


Laser Capture Microdissection Forces

For successful LCM, Tissue-Activated Film force must be greatest.


SEM Image of LCM pulses


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.

Proper Film contact No Film contact


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 smaller#’s 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


1. Prepare tissue 2. Locate cells 3. Place cap

4. Pulse laser 5. Remove cap 6. Extract molecules

The Laser Capture Microdissection Process


Microdissect a Variety of Samples

Microdissect any morphological shape

•Microdissect single cells or multi-cellular structures—regardless 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.


ArcturusXT™ Microdissection Instrument


ArcturusXT Software Graphical User Interface

1. Load Materials

2. Inspect Samples

3. Select Cells

4. Microdissect

5. QC


Large Area Captures

Laser Cutting vs. LCM: Choice based on Experimental Objective

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

UV Laser CuttingIR Laser Capture Microdissection

Large Captures

~650u areas


Laser Cutting vs. LCM: Choice based on Experimental Objective

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

dissections

Small Area Captures

UV Laser CuttingIR Laser Capture Microdissection

Small Captures

~30u areas


Phase Contrast DIC

Cultured TM3

Cells

Chinese hamster ovary (CHO)

cells

Cultured TM3 cells

Cultured 3T3 cells

ArcturusXT – Phase Contrast and DIC

Ideal for unstained samples


ArcturusXT – Fluorescence

Triple labeled bovine pulmonary artery endothelial (BPAE) cells.

Mitochondria = red, F-actin=green and Nuclei= blue. Visualized

simultaneously using an Omega triple band dichroic filter.

Human Breast Carcinoma, anti-

cytokeratin/Cy3

Cultured HELA cells exposed to

BCECF, a cytoplasmic pH indicator.


Single Cell Capture - ArcturusXT™ IR Laser Capture Microdissection

Frozen Mouse Brain –

Histogene Stain

Before LCM

After LCM

CapSure


FFPE Cresyl Violet - Stained

Human Bone Marrow

ArcturusXT™ UV Laser Cutting

Before Laser Cutting

After Laser Cutting

CapSure


ArcturusXT LCM – FFPE tissue section

Rat FFPE kidney section.

Paradise stain.

Before LCM

After LCM

CapSure Cap


Drosophila Embryo X-gal-

Stained Fluorescence

Before Laser Cutting

After Laser Cutting

CapSure

Fluorescence Dissection - ArcturusXT™

UV Laser Cutting


ArcturusXT™ - Microdissected GFP Neurons


ArcturusXT laser cutting – live plant microdissection

Whole mount preparation.

Blade of grass on frame

membrane slide.


Multiple Stage Formats

• Large Slide Stage Insert

> 25mm, 38mm and 50mm slides

> Great for neuroscience applications

• Petri Dish Stage Insert> accommodates 50mm x 7mm Petri dishes

> Easy swap out for live cell or tissue based

applications

− Live cell imaging

− Live cell microdissection

ArcturusXT


ArcturusXT Live Cell Microdissection


Cell Identification

HistoGene®

Kits

Cell Isolation and

Extraction

PicoPure® Kit (frozen)

Paradise®

PLUS Kits (FFPE)

Amplification

RiboAmp® PLUS

Kit (frozen)

Paradise® PLUS

Kit (FFPE)

Labeling

Turbo

Labeling™

Kit

Cell Selection

ArcturusXT™

LCM

System

Downstream

Analysis

Integrated Systems for Microgenomics®

Tissues preparation

王森右Field Applications Scientist

• Tissue Preparation

• Tissue Sectioning

• Staining


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


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.


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 Zap or similar product to clean equipment


Laser Capture Microdissection Forces

For successful LCM, Tissue-Activated Film force must be greatest.


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


Protocol for Freezing Tissue

Specimen Freezing with Isopentane

1. Add a thin layer of OCT to bottom of cryomold

2. Place tissue specimen on top OCT layer

3. Add additional OCT to cover specimen

4. Place cryomold into cooled isopentane to freeze

5. Once tissue block is completely frozen transfer to dry ice or to

a –70C freezer for storage


Frozen Tissue Sectioning

Gloves must be worn at all times

Cryostat must be cleaned prior to use. All surfaces must be wiped

down with 95-100% ethanol, especially knife holder and anti-roll plate Recommended Section thickness

LCM alone = 8-10umLC+LCM = up to 100um

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


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


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


Preparation of Tissue Sections

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.


Optimal Staining vs. RNA Quality

RNA QualityVisualization


Staining and Dehydration

• Staining

− 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 Removal of water inactivates nucleases

> Graded ethanol series – 75%, 95%, 100%

> Ethanol concentrations must be freshly prepared

> Xylene steps performed in fume hood

> Air Drying no more than 5 minutes in a fume hood

− Provides conditions that maximizes the LCM process


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


Histochemical Staining

HistoGene Stain for Frozen Tissue

• 75% ETOH - 30secs

• NF dH20 - 30 secs

• Histogene stain -10-30 secs

• NF dH20 - 30 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 Stainfor FFPE Tissue Xylene – 2-3 mins

Xylene _ 2-3 mins

100% ETOH – 1 min

95% ETOH – 1min

75% ETOH – 1 min

NF dH20 – 30 secs

Paradise Stain – 30-45 secs

NF dH20 – 30 secs

75% ETOH – 30 secs

95% ETOH – 30 secs

100% ETOH – 1 min

Xylene – 5 mins

Total Time = 18 mins

Aqueous Time = 7 mins


HistoGene® Immunofluorescence Kit

biotin

tissue antigen

1o Antibody

streptavidin

Cy3

Total staining time = 17 mins


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


RNA Quality Assessment of Tissues

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)

Frozen Tissues: Arcturus Protocol #1-Tissue Scrape Protocol for

Verifying RNA Quality

FFPE Tissues: Paradise® Plus or Paradise® Plus WT-RT Kits


Slide Formats for LCM and LC/LCM

Standard Glass

Glass Membrane

Frame Membrane

•Multiple modes for microdissection.

•Enables flexibility w/ applications.

•Removes dependence on tissue dehydration.


Compatible with ALL standard glassand membrane slides

Standard Glass Slides Glass Membrane Slides Frame Membrane Slides

CapSure

TissueGlass Slide

IR Laser LCM

UV Laser Ablation

IR Laser LCMIR Laser LCM

CapSureCapSure

TissueTissue

UV Laser Cutting and Ablation

UV Laser Cutting and Ablation

MembraneSlide

Membrane

Frame

Glass Slide

LCM & UV Ablation LCM, LCM & UV Ablation LCM & UV Ablation


Cell Quantities and Downstream Applications

Source: Espina V etal, ―Laser-Capture Microdissection‖, Nature Protocols (1:2), 2006

http://www.nature.com/nprot/journal/v1/n2/pdf/nprot.2006.85.pdf


Arcturus Protocols & App Notes


Q&A For Research Use Only.

Not intended for any animal or

human therapeutic or diagnostic

use.

Arcturus XT Microdissection Systemweb.ecologia.unam.mx/labmicrolas/documentos/6... · 6 | Life Technologies | 11/8/2012 Introduction to LCM Microgenomics Quantitative genomic and

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