Microsoft PowerPoint - 20090716 Proteomic Application of LCM ()v2.ppt: 2009716
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Obtaining Pure Cells By Laser Obtaining Pure Cells By Laser MicrodissectionMicrodissection
Identify Target Cells Isolate Target CellsVisualize Biopsies
Normal
ADH
DCIS
IDC
See What You've Been Missing See What You've Been Missing
Gene 1
Proteoglycan 1
Gene 2
CD53 antigen
Gene 3
Proteosome subunit
Mixed Pure
Requires patience and dexterity.
Difficult to microscopically verify the purity of the cells and the accuracy of the dissection.
In 1970In 1970’’ss
Histochem J., Vol 8, Issue 4, 387 - 401, 1976
The laser in the Lowry technique for microdissection of freeze-dried tissue slides.
UV Laser Cutting Technology for Microdissection
LMD LMPC
Since 1996Since 1996
Science, Vol 274, Issue 5289, 998-1001, 1996 Science, Vol 278, Issue 5342, 1481-1483, 1997
National Institutes of Health & National Cancer Institute
LCM Caps
Frozen Mouse Brain – HistoGene Staining
Before LCM
After LCM
ArcturusXT™ microdissection
CapSure
Tissue
Large Area  Captures 
Small Area  Captures
CapSure
Tissue
IR Laser LCM IR Laser LCM
CapSure CapSure
Standard Glass Slides Membrane Slides Membrane Framed Slides
Membrane Slide
Frame slide
Glass Slide
Capture cells
Isolationextraction
Amplification & labeling
Pre-LCM
from LCM Captured Cellsfrom LCM Captured Cells
LCM
Sequence
Courtesy of Dr. Lin Luo and Dr. Mark Erlander, R. W. Johnson Pharmaceuticals
LCM of adjacent cells
Large Dorsal Root Ganglion Rat Neurons, Nissl stained
Adjacent cells can be collected because they are not ablated or damaged during collection
Undamaged cells remain
QC QC
Human Blood Smear
Before Laser Cutting
After Laser Cutting
RNA Yield: 2.7 ng/μl RIN: 9.5
RNA Yield: 2.6 ng/μl RIN: 9.6
RNA Yield: 3.1 ng/μl RIN: 9.7
Microdissection does not affect cell or nucleic acid viability
A transcriptome atlas of rice cell types uncovers cellular,functional and developmental hierarchies
Nature Genetics 41, 258 - 263 (2009)
METHODS Laser microdissection: Cells were isolated by microdissection with Veritas and Pix-Cell IIe Microdissection Systems (Arcturus/Molecular Devices).
PNAS, 2005, 102: 8036-8041
Gene Expression Profiling of LongGene Expression Profiling of Long--Range Range Projection NeuronsProjection Neurons
Relative expression levels of UCHL1 in zebra finch HVC
Journal of Virology (2005) 79(22):14079–14087
Quantification of Latent HSV-1 and VZV at the Single-Cell Level
LCM of LAT+ and LAT- neurons
Neurons
Developmental Biology 312 (2007) 596–612
Gene expression analysis of Gene expression analysis of zebrafishzebrafish retinal ganglion cellsretinal ganglion cells
Gene ontological analysis of changes in gene expression in RGCs during optic nerve regeneration
Identifying Cell-Specific Expression Signatures
Molecular Signatures of Cancer ProgressionMolecular Signatures of Cancer Progression
Ma, Xiao-Jun et al., PNAS 100(10): 5974-5979, 2003.
Applications
Journal of Biological Chemistry 279: 37061-37066 (2004)
LCM of a plaque region from thioflavin-S-stained AD brain section
Identification of proteins enriched in the plaque regions by LC/MS
Protein Microarrays
Nature Reviews_Drug Discovery 1: 683-695
LCM + MALDI MSLCM + MALDI MS
1. LCM to get specific target cells
2. Transfer firm with cells is removed from LCM caps and placed on MALDI plate
3. Apply Matrix
Protein Microarrays
FH (Benign follicular hyperplasia) FL (Follicular lymphoma)
Clinical Cancer Research 11: 5847-5855 (2005)
Normal cell
Molecular changes
Year 10
Year 6
Year 3
Year 0
Cancer cell
http://cgap.nci.nih.gov/Tissues/LCM
In 1996, the Cancer Genome Anatomy Project (CGAP) began. This program of the National Cancer Institute (NCI) is studying the molecular changes that occur when a normal cell is transformed into a cancer cell.
LCM Web Seminar Series
1. Enhanced interferon signaling pathway in oral cancer revealed by quantitative proteome analysis of microdissected specimens using 16 O/ 18 O labeling and integrated 2DLC-ESI-MALDI tandem MS
Mol. Cell. Proteomics 2009 Mar 18.
2. Immunohistochemical and Biogenetic Features of Diffuse-Type Tenosynovial Giant Cell Tumors:The Potential Roles of Cyclin A, P53,a nd Deletion of 15q in Sarcomatous Transformation
Clin Cancer Res 2008;6023 14(19) October 1,2008
3. Heat Shock Protein 90 Overexpression Independently Predicts Inferior Disease-Free Survivalwith Differential Expression of the A and B Isoforms in Gastrointestinal Stromal Tumors
Clin Cancer Res 2008;14(23) December 1, 2008
4. Expression and localization of Tmie in adult rat cochlea Histochem Cell Biol (2008) 130:119–126
5. Increase of disintergin metalloprotease 10 (ADAM10) expression in oral squamous cell carcinoma
Cancer Letters 245 (2007) 33–43
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