Laser Capture Microdissection
Study of gene and protein expression in anatomically complex organs is difficult because of tissue heterogeneity; the organ is 'contaminated' by admixture of many different cell types. Functional genomics or proteomics can now be performed at the level of individual cells in a tissue, allowing more complete analysis of mechanisms of disease in situ. Laser Capture Microdissection (LCM) uses a laser beam to isolate and transfer targeted cells, for isolation of DNA, RNA or protein. The technique has been used to detect mutations and measure gene expression and protein levels in normal, cancerous, injured, or fibrotic tissue. Targeting of specific cells in the kidney is difficult using routine morphologic stains. Immunofluorescent-LCM allows identification and targeting of specific tissue cells for LCM without mRNA loss. We will review LCM and how it has been coupled to genomic and proteomic techniques that might enhance our understanding of human renal diseases, including glomerulonephritis and acute renal failure, whose pathophysiology have been mysterious. LCM can be used under conditions that are routinely used for renal biopsies, ie, formalin fixed paraffin embedded material for DNA analysis, and frozen sections for mRNA analysis. LCM dissected material can serve as the input material for a wide range of assays including analysis of DNA mutations, gene expression, protemics, and even single cells (Table). We have found these techniques to be extremely helpful in pinpointing expression of newly discovered genes in the kidney. However, extreme care must be taken to insure the that microdissected cells are taken from the correct tubule segment. These techniques might enhance our diagnostic abilities by providing a molecular diagnosis from a single injured glomerulus or renal tubule.

Table 1: Analysis of LCM samples

Detection	# spots	Single Gene	Many Genes	References
DNDNA	1	LOH		(1)
	1	gene mutations		(2)
mRNA	1	RT/PCR		(1;3)
	few	qRT/PCR		(4-7)
	1000+		cDNA libraries	(8-10)
	1000+		cDNA microarrays	(4;8;11)
Protein	250-2000	Western blotting		(12-15)
	50	ELISA		(16)
	7,000-30,000		2-D gels	(11;11;13-15)
	25-500		SELDI/TOF	(17;18)
	2,000+		Protein arrays	(19)
	2,000+		Antibody arrays	(18)

References

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2. Fend,F. , Quintanilla-Martinez,L. , Kumar,S. , Beaty,M.W. , Blum,L. , Sorbara,L. , Jaffe,E.S. , and Raffeld,M. 1999. Composite low grade B-cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas - A molecular analysis using laser capture microdissection . American Journal of Pathology 154 :1857-1866.
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6. Wong,M.H. , Saam,J.R. , Stappenbeck,T.S. , Rexer,C.H. , and Gordon,J.I. 2000. Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection . Proc.Natl.Acad.Sci.U.S.A 97 :12601-12606.
7. Specht,K. , Richter,T. , Muller,U. , Walch,A. , Werner,M. , and Hofler,H. 2001. Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue . Am.J.Pathol. 158 :419-429.
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16. Simone,N.L. , Remaley,A.T. , Charboneau,L. , Petricoin,E.F., III, Glickman,J.W. , Emmert-Buck,M.R. , Fleisher,T.A. , and Liotta,L.A. 2000. Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection . Am.J.Pathol. 156 :445-452.
17. Paweletz,C.P. , Liotta,L.A. , and Petricoin,E.F., III. 2001. New technologies for biomarker analysis of prostate cancer progression: Laser capture microdissection and tissue proteomics . Urology 57 :160-163.
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19. Paweletz,C.P. , Charboneau,L. , Bichsel,V.E. , Simone,N.L. , Chen,T. , Gillespie,J.W. , Emmert-Buck,M.R. , Roth,M.J. , Petricoin III,E.F. , and Liotta,L.A. 2001. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front . Oncogene 20 :1981-1989.