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From the Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Processing of Tissue
Five-micron histological sections of formalin-fixed,
paraffin-embedded tissue were placed on uncharged, noncoated glass
slides. After drying, slides were deparaffinized (4 changes of xylene,
5 minutes each; 3 changes of 100% ethanol, 1 minute each), rinsed in
distilled water, and stained as described below. Five-micron
histological sections of frozen tissue were also placed on uncharged,
noncoated glass slides, immediately fixed in 70% ethanol for 30
seconds, and stained as described below. From each specimen block
(frozen and fixed), serial sections were used for each different stain
and for the LCM versus manual tissue dissection, so that
within-specimen variability due to tissue heterogeneity was minimized.
After staining, all sections were rinsed in distilled water, dehydrated
in three changes of absolute ethanol (1 minute each) followed by four
changes of xylene (5 minutes each). The slides were then air-dried and
used for manual dissection or microdissection.
Staining of Tissue
Filtered, aqueous solutions of toluidine blue, azure B, and methyl
green (Sigma, St. Louis, MO) were used for staining. Initial
experiments varying dye concentrations and staining times established
the concentrations and times that gave morphologically satisfactory
results. Paraffin-embedded tissue sections were stained in 0.01%
toluidine blue for 2 minutes, 0.5% azure B for 30 seconds, 0.5%
methyl green for 30 minutes, or Mayer’s hematoxylin solution (Sigma)
for 2 minutes. Frozen tissue sections were stained in 0.1% toluidine
blue for 30 seconds, 0.5% azure B for 30 seconds, 0.5% methyl green
for 30 minutes, or Mayer’s hematoxylin solution for 30 seconds.
Staining by Mayer’s hematoxylin was followed by immersion in bluing
solution (Richard-Allan Scientific, Kalamazoo, MI) for 30 seconds. For
unstained controls, sections were treated identically
(deparaffinization, hydration, and dehydration) to the stained slides,
except that they were immersed for 30 seconds in deionized water in
place of dye solution.
LCM and Manual Dissection of Tissue
LCM was performed with a Pixcell II Laser Capture Microscope
(Arcturus Engineering, Mountain View, CA) in the Alvin J. Siteman
Cancer Center Tissue Procurement Core Facility. Tissue (5 µm
thickness) obtained from an average of 500 firings of a 30-µm laser
spot size was captured on the thermoplastic caps and digested in 20
µl proteinase K buffer. Hence, the digestion proceeded at a density
of approximately 8.5 x 104
µm3 (= 8.5 x
10-5 µl) of tissue
volume per 1 µl of digestion buffer. Based on visual examination of
the transfer films and the tissue sections after cell transfer, one
laser pulse corresponded to approximately five to six cells. This
number varied somewhat, depending on the tissue dissected. In general,
however, a total of 2500 to 3000 procured cells were represented in the
20 µl of digestion buffer. For each case, the same area of each
differently stained serial section was subjected to LCM, so that the
cell population for a single specimen across different staining
procedures was equivalent. Tissue obtained by manual dissection was
sampled by scraping the entire tissue section from the slide with a
razor blade, followed by placing the tissue into proteinase K buffer,
at an approximate ratio of 1 square cm tissue area per 100 µl of
buffer. This corresponded to a density of approximately 5 x
106 µm3 (= 5 x
10-3 µl) tissue volume
per 1 µl of digestion buffer. Thus, the microdissected tissue was
digested at a much lower ratio of tissue to digestion buffer than the
manually dissected tissue. Nevertheless, for the latter there was still
an approximately 200-fold excess of digestion buffer volume over tissue
volume.
DNA Preparation
The tissue sampled by LCM or by manual dissection was digested
overnight at 60°C in proteinase K buffer. The composition of
proteinase K buffer was as follows: 10 mmol/L Tris/HCl, pH 8.0, 1
mmol/L EDTA, 1% Tween 20, and 0.04% (w/v) proteinase K (Boehringer,
Indianapolis, IN). Undigested debris was subsequently removed by
centrifugation at 10,000 x g for 5 minutes, and
proteinase K was inactivated by incubation at 95°C for 10 minutes.
For dilution experiments, tissue lysates from manually dissected
specimens corresponding to 5 x 106
µm3 tissue volume per 1 µl of digestion
buffer (see above) were further diluted in digestion buffer at 1:5,
1:25, and 1:125 after heat inactivation. Lysates were used for DNA
analysis by TaqMan PCR or by conventional PCR without further
purification and stored at -70°C for up to 2 weeks without loss of
amplifiable DNA.
DNA Analysis by TaqMan
Quantitative analysis of genomic ß-actin DNA was performed by
monitoring in real-time the increase in fluorescence of the dye SYBR
green I (Molecular Probes, Inc., Eugene, OR) as described5
using the TaqMan instrument (ABI Prizm 7700 Sequence Detection System,
Perkin Elmer, Norwalk, CT). The sequences of the primers used for
amplification of ß-actin DNA were 5'-CGAGAAGATGACCCAGATCATGTT-3' and
5'-CCTCGTAGATGGGCACAGTGT-3'. ROX was used as the reference dye. All
reactions were repeated in duplicate. The instrument was calibrated
using human genomic DNA standards of known concentration as determined
spectrophotometrically at 260 nm wavelength. Selected results obtained
by TaqMan amplification were confirmed semiquantitatively by
conventional PCR using the same primers, and the amplification product
of approximately 150 bp was detected on a 2% agarose gel. All
amplification curves generated with TaqMan from stained tissue had the
typical sigmoidal appearance of amplification curves generated from
unstained tissue, which indicates that traces of these dyes do not
interfere with the fluorescence detection system. As noted by
others,4
eosin, a cytoplasmic dye which we had initially
included in the study, resulted in highly atypical TaqMan amplification
curves due to its innate fluorescence, and we have therefore excluded
this dye from this study.
Statistical Analysis
Data sets were analyzed for statistical significance by Student’s
t-test in the two-tailed form. Significance was defined as
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TaqMan PCR was used next to compare the effect of the histological dyes
on retrieval of amplifiable DNA. Overall, there was considerable
variability in the amount of amplifiable DNA recovered from
approximately equal amounts of manually dissected tissue. Specimens
from archived tissue blocks could not be controlled for equal fixation
conditions. This could be one source of interspecimen variability,
although prospectively collected, snap-frozen tissue specimens also
demonstrated variability in recovery of amplifiable DNA. Despite
attempts to select specimens displaying similar degrees of cellularity
and necrosis, another obvious source of this variability is simply the
interspecimen cellular heterogeneity. For formalin-fixed,
paraffin-embedded tissue harvested by manual dissection, amplification
efficiency was approximately equal irrespective of the staining
procedure used, although there was a tendency (not statistically
significant) for methyl green to yield DNA with increased amplification
efficiency (Figure 1A)
. The type of stain used had a greater effect in frozen tissue
harvested by manual dissection (Figure 1B)
. Amplification was
consistent and high after staining with azure B, toluidine blue, and
methyl green, but was statistically significantly lower after
hematoxylin staining or when no stain was used. In our experience, and
in accordance with previous reports,2, 3, 4
the results with
hematoxylin-stained tissue obtained by manual dissection were
essentially irreproducible. That is, we observed that tissue initially
yielding no amplifiable PCR product gave a satisfactory result when
repeated on independently sectioned and stained tissue, and vice
versa. In this respect, the data with hematoxylin-stained tissue
presented in Figure 1
reflect our experience at one given point in
time. In contrast, we did not observe this degree of unreliability with
the other dyes.
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, and conventional PCR
followed by visual inspection of ethidium bromide-stained bands
separated electrophoretically on agarose gels (not shown). Therefore,
it is unlikely that this effect represents an artifact generated by
interference of the histological dyes with the TaqMan detection system.
Another possible explanation for the result is that the hematoxylin dye
or some other component of the cell lysate is inhibiting PCR
amplification. To test this hypothesis, fivefold serial dilutions of
cell lysates from hematoxylin-stained, manually dissected tissue were
generated. In duplicate reactions of two independently stained
sections, cell lysate at a concentration of approximately 5 x
106 µm3 tissue/µl of
digestion buffer (comparable to that in the first set of experiments)
again yielded no amplifiable DNA. A subsequent fivefold dilution
of these lysates resulted in a 200- to 1000-fold increase in the amount
of amplifiable DNA, while further fivefold dilutions demonstrated
corresponding three- to fourfold decreases in amplifiable
material (data not shown). In summary, these data suggest that the
effect of hematoxylin-stained tissue on subsequent TaqMan PCR
amplification is inhibitory and that this inhibition can be alleviated
when the concentration of the lysed tissue falls below 1 x
106 µm/µl of lysis buffer.
For tissue isolated by LCM, DNA amplification was approximately equal,
irrespective of the staining procedure, for both paraffin-embedded and
frozen tissue (Figure 1 C and 1D)
. In particular, hematoxylin was as
reliable a stain as methyl green, in contrast to the results obtained
with manually dissected tissue.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The objective of this study was to investigate the usefulness of
different nuclear dyes for microdissection of histological tissue with
subsequent amplification of DNA by TaqMan PCR. Both formalin-fixed,
paraffin-embedded tissue and frozen tissue were sampled from
histological slides either by manual gross dissection or by LCM. The
choice of the dye had a marked influence on the outcome of DNA
amplification when the entire tissue section was manually sampled from
the histological slide. Such sampling, followed by PCR amplification,
is commonly done in parallel to microdissection as a quality control to
make certain that the content of amplifiable DNA in the tissue has not
been unacceptably reduced by autolysis or the staining procedure. Our
results show that in this situation, sampling of hematoxylin-stained or
unstained tissue would in many instances give false negative,
misleading results, whereas staining with methyl green, azure B, or
toluidine blue O allows reliable assessment of the DNA content of the
tissue (Figure 1 A and B)
.
The unsatisfactory amplification of DNA from manually sampled entire
tissue sections stained by hematoxylin has been reported
previously.2, 3, 4
In our hands, the average DNA
amplification was less after staining with hematoxylin than with other
nuclear dyes (Figure 1 A and B)
, and TaqMan or conventional PCR failed
to yield an amplification product in a substantial number of reactions.
Any of the multiple chemical ingredients of the hematoxylin staining
solution, which could inhibit the proteinase K digestion or the PCR
reaction, may be responsible for this effect. As to the lack of
amplifiable DNA from unstained frozen tissue, the mechanism of this
effect is unclear. It is not likely due to a washout of tissue
inhibitors by the staining solutions, as unstained slides were
subjected to similar hydration/dehydration steps. Rather, it may be
that the staining solutions, when not inhibitory to the Taq
polymerase, render the genomic DNA more accessible, either by releasing
it from bound nucleoprotein complexes or chemically cleaving it into
smaller fragments that are more readily released from the tissue. This
latter hypothesis is partially supported by the finding that
unstained tissue from formalin-fixed specimens generally yielded
more amplifiable DNA than that from frozen sections.
A previous report has found that after sampling entire tissue sections
from the slides by manual dissection, staining with methyl green
apparently enhanced DNA recovery relative to unstained
tissue.2
We have made a similar observation (Figure 1A)
,
although the difference was not statistically significant. We were
surprised, however, that the enhancement by methyl green was
particularly pronounced and statistically significant when frozen
tissue samples were examined (Figure 1B)
. The mechanism that leads to
this enhancement is not entirely clear and may be multifactorial. Since
cationic dyes such as methyl green bind to DNA, it is conceivable that
this binding enhances the digestion of DNA-bound proteins by proteinase
K, or increases the efficiency of PCR by enhancing the accessibility of
the DNA during primer hybridization or extension.
In marked contrast to the situation where entire tissue sections
are manually sampled, there was no influence of the staining method on
DNA amplification when tissue was sampled by LCM (Figure 1 C and D)
.
Most likely, this difference is related, at least in part, to the
amount of tissue sampled. The tissue pieces obtained by LCM are quite
small, resulting in little carryover of dye substances into the
proteinase K digestion solution and the PCR reaction. Consequently,
inhibitory ingredients of the hematoxylin stain, or enhancing dye
substances such as methyl green, will be present in concentrations too
low to produce any effect on tissue digestion or DNA amplification.
The main finding of this study is that DNA recovery from manually dissected tissue is not predictive of the recovery after LCM sampling from the same tissue. From our results on tissue sampled by manual dissection, we would have predicted that hematoxylin is an unsuitable stain for microdissection, whereas in fact it gave satisfactory results in the actual LCM experiments. Thus, based on the experience of 16 cases used in this study, hematoxylin can be recommended for LCM-based microdissection.
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Footnotes |
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Supported by Department of Defense grant DAMD 17–99-1–9026 (to J. M.), National Cancer Institute grant K08CA8790101 (to S. A. A.), and the Siteman Cancer Center.
Accepted for publication November 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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