Laser Capture Microdissection
(LCM) is a method for obtaining pure populations of cells from
heterogeneous samples. LCM permits the selection and capture of
cells, cell aggregates, and discrete morphological structures from
thin tissue sections. For LCM, frozen and OCT embedded, or
formalin-fixed and paraffin embedded tissue sections are processed,
stained, and dehydrated, then scanned under an inverted microscope.
Cells are visualized through a thermoplastic film which is attached
to the bottom of an optically clear microfuge tube cap. A laser
pulse, directed onto the target cells through the film, melts
the film and allows it to flow onto the targeted area where it cools
and bonds with the underlying cell(s). The film including the
adhered cells or clusters is then lifted. Captured cells can then be
used for nucleic acid studies, including SNP analysis, endpoint and
real-time RT-PCR, and mRNA expression profiling.
Formalin vs. Frozen
LCM is usually carried
out on frozen sections when samples will be used for RNA isolation.
This is because formalin fixation introduces crosslinks between
nucleic acids and proteins, resulting in fragmentation of the RNA.
Freezing avoids crosslinking but introduces a different problem.
Endogenous nucleases are not irreversibly inactivated, and can
become active during the tissue processing steps (fixation,
staining, destaining, and dehydration) carried out after sectioning.
An Improved Protocol for Staining
Ambion scientists performed a detailed analysis of
RNA recovered from LCM sections after various steps of the conventional
post-sectioning processing protocols for frozen samples. These studies
revealed that significant RNA degradation occurred during exposure of
the section to aqueous solutions, presumably due to reactivation of endogenous
nucleases. To circumvent this problem, Ambion developed a staining procedure
that avoids exposing the sections to water at any step. (See our LCM
Staining Kit.) Using the optimized "water-free" tissue processing
protocol in conjunction with the RNAqueous-Micro Kit, total RNA of excellent
quality, as assessed by microfluidic capillary electrophoresis and real-time
RT-PCR, can be obtained from pure populations of cells selected by LCM
Figure 1. Real-Time
RT-PCR of ICM Samples. Mouse spleen
(A) and liver (B) samples (1 mg, 3 mg and 10 mg) were dissected by LCM
and RNA was isolated using the RNAqueous-Micro Kit according
to protocol. The RNA was then used as a template for the amplification
of GAPDH by real-time RT-PCR.
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LCM Staining Kit
Reagents for processing 80 slides
. Maintaining High Sensitivity when Working with Small-Volume Samples2
. Isolate and Analyze Total RNA from Cells Harvested by Laser Capture
. Laser microdissection and nucleic acid purification - a Leica - QIAGEN
. Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions5
. Laser Capture Microdissection (LCM) and Expression Profiling of Long-Range Projection Neurons6
. Differential Gene Expression Analysis of Pure Cell Populations from Frozen UterineTissue Using Laser Capture Microdissection (LCM) and cDNA Microarrays7
. Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR8
. Laser Capture Microdissection of Cells Labeled with Enhanced Green Fluorescent Protein9
. Quantitation of the Protein MIA as a Marker for
Chondrocytes in Research Samples10
. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis11
. Preserve Samples for RNA Expression Microarrays RNAlater Around the World