42
Chromosome, Microdissection and Microcloning
(a)
(b)
Fig. 6
(a) Arturus PixCell II
Laser-capture Microdissection System
that consists of an Olympus inverted
microscope with 4
×
,10
×
,20
×
,and
40
×
objectives, near-infrared diode
laser, user-selectable 7.5 mm, 15 mm,
and 30 mm spot sizes, CCD color
camera and monitor, image-archiving
workstation. (b) Close-up at the laser
beam to capture a single cell.
on TaqMan polymerase chain reaction
amplifcation oF genomic DNA prepared
From fxed and Frozen tissue For laser-
capture microdissection. They concluded
that DNA recovery From LCM-sampled
tissue is independent oF the histological
stain chosen to highlight nuclear detail.
The use oF immunohistochemical stains
allows the selection oF cells according to
phenotypic and Functional characteristics.
Depending on the starting material, DNA,
good quality mRNA, and proteins can
be extracted successFully From captured
tissue Fragments.
More recently, LCM has been refned to
c
ap
tu
r
es
ing
l
ec
e
l
l
sinasp
e
c
im
en
.Th
e
capture oF individual cells with a new
NIH LCM microscope epi-irradiates the
EVA polymer overlying individual cells
with 1-ms laser pulses Focused to 6
µ
m.
A computer-controlled arm precisely po-
sitions a 40-
µ
m-wide strip oF a cylindrical
EVA surFace onto a sample with a light
contact Force (ca. 0.1 g). The small contact
Force and contact area on the flm on the
sample diminishes nonspecifc transFer to
negligible levels.
With the advent oF LCM, cDNA libraries
are being developed From pure cells ob-
tained directly From stained tissue, and
microhybridization arrays oF thousands oF
genes can now be used to examine gene ex-
pression in microdissected human tissue
biopsies oF various specifc pathological
lesions, especially malignant neoplasms.
Similarly, the use oF LCM is suFfcient
to isolate nanogram quantities oF high-
quality RNA. Together with the use oF
several internal standards and microcap-
illary electrophoresis oF input RNA, two
rounds oF linear molecular amplifcation
have been used to generate suFfcient quan-
tities oF labeled target For hybridization
to high-density oligonucleotide expres-
sion arrays. Results demonstrate that the
technique is reproducible and generates
only modest biasing oF the original tran-
script population.
±or Further details regarding applica-
tions oF LCM, the reader is encouraged
to reFer to the review article by ±end
and RaFFeld. It is an excellent source
For determining the pros and cons oF
LCM versus conventional microdissection
methods, and the limitation oF technol-
ogy. The review also discusses the cur-
rent applications with reFerences to DNA
analysis, gene expression analysis, global
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