Cancer research - Isolating cell areas from human lung carcinoma from cryosections
Axel Niendorf and Michael Harsch, MEDEEA Forschungs-GmbH, Hamburg, Germany
Introduction
The characterization of molecular
genetic changes of specific cell
populations is important for the
understanding of a developing
tumor. When analyzing total tumor
tissue, one faces the problem that
molecular characteristics, for example,
the expression of specific
mRNAs, might be diluted. A rapid
one-step microdissection technique
applied for the isolation of tissue
areas has been developed. The special features of the Eppendorf MicroDissector consist of an ultrasonically
oscillating metal needle
(MicroChisel) and a piezo-driven
micropipette (Fig.1).The validity of
this technique was demonstrated in
human lung large-cell carcinoma by
real-time quantitative reverse transcriptase-
polymerase chain reaction
(RT-PCR) assays of the intermediate
filament vimentin, the proliferationassociated
antigen cyclin D1, and
carcinoembryonic antigen (CEA)
after linear RNA amplification [1].
Materials and methods
Tissue preparation
Parallel blocks of fresh tumor tissue
were fixed in a buffered 4 % formaldehyde
solution and embedded in
paraffin or shock-frozen [1,2] in
liquid nitrogen and stored at -80 °C
until further analysis.
Paraffin sections were used for control
experiments by immunostaining.
For the purpose of microdissection,
a microtome was used to obtain 10
µm sections from the frozen blocks
which were then mounted on glass
slides. The sections were immediately
hematoxylin and eosin-stained
with special care to obtain high quality
RNA and dehydrated in graded
alcohol and xylene (10 seconds
each). All solutions were prepared
with diethyl-pyrocarbonate-treated
H20 (DEPC 0.1 %). They were kept
in Falcon tubes and pipetted separately
onto the slides. To improve RNA
recovery, slides of uncovered cryosections
were stored at 4 °C on silica
gel in an exsiccator [1].
Devices
• Eppendorf MicroDissector (Fig. 2)
including a cutting tool and an
electronic micropipette (Fig. 1)
• Inverted microscope with
2 Electronic Micromanipulators
TransferMan NK2 (Fig. 3)
Consumables
• Eppendorf MicroChisel
• Eppendorf Filtertip MDS,
• Eppendorf pipette tips,
PCR clean
• Eppendorf 0.5 ml tubes,
PCR clean
Microdissection procedure
Two micromanipulators were used to
move both the cutting tool with
MicroChisel and the micropipette.
During dissection the cryosections
were coated with approx. 15 µl xylene.
For analysis the tissue is fragmented
into subcellular particles by the
oscillating MicroChisel. The xylene
containing the material of interest is
aspirated into the Filtertip MDS.
After evaporation of the xylene, total
RNA is isolated from the cell fragments
and used for further experiments.
RNA amplification
Total RNA was dissolved in 5 μl of
diethyl-pyrocarbonate-treated H20
(DEPC 0.1 %) and enzymatically
processed with DNase I and RNase
inhibitor [1].
cDNA synthesis using an oligo-dT
primer with an additional T7 promoter
sequence and subsequent RNA
amplification has been described in
[Barry C, Pat Brown Lab: Modified
Eberwine "antisense" RNA amplification
protocol (http://cmgm.stanford.
edu/pbrown/protocols/ampprotocol_
2.txt)]. For protocol modifications
see [1].
RT-PCR
First-strand cDNA was prepared [1]
and aliquots were analyzed by realtime
PCR with a LightCycler instrument
[3] using the primer pairs described
in [1].
Data analysis
The LightCycler software was used
for the analysis. PCR product accumulation
was determined by measuring
the fluorescence once per cycle
at the end of the extension phase to
generate an amplification curve for
each sample [1].
Conclusion and discussion
The use of the oscillating
MicroChisel allows precise microdissection
of cells for analysis and their
easy detachment from the glass
slide.
The time necessary for preparation
will vary depending on the number
of cells cut and the tissue architecture.
During the preparation of larger
areas the xylene pool became enriched
by the tissue particles.
Remaining tissue particles can be
removed under visual control by
repeated covering and aspiration
with xylene and pooled together with
the initial sample [1].
Preparation with the oscillating
MicroChisel allows a sharp separation
between the dissected area and
unwanted tissue that remains intact
for further analysis.
Analysis of pure cell populations is a
prerequisite in the study of differential
gene expression in complex tissues,
especially with regard to
advanced molecular techniques [1].
It is possible to conduct subsequent
investigation in any standard down
stream application with tissue obtained
by the Eppendorf MicroDissector
method [1]. It can be applied to
either paraffin sections or unfixed
cryostat sections that are often
recommended for mRNA extraction.
It is a general problem of microdissection
that dry non-coverslipped
slides do not allow fine cytological
detail. The preparation under xylene
provides excellent optical conditions.
In addition, in contrast to aqueous
buffer solutions, the tissue does not
suffer variable adherence to the
glass slide, which can diminish the
precision of dissection [4, 5]. RNA
stability is also improved in an anhydrous
environment.
The validity of this technique is
demonstrated by the RT-PCR [1].
The quantitative analysis [1] is in
accordance with immunohistochemistry.
Microdissection of immunophenotypically
defined cell populations allows
cell-specific mRNA analysis according
to antigen expression [6, 7].
References
[1] Harsch et al. 2001: A new method for histological microdissection utilizing an ultrasonically oscillating MicroChisel.
Demonstrated by differential mRNA expression in human lung carcinoma tissue. American Journal of Pathology,
Vol. 158, 6: 1985–1990.
[2] Current Protocols 2002: Discovery and analysis of differentially expressed genes in single cells and cell populations.
25A.1 includes basic protocols for the preparation of frozen sections and fixed paraffin-embedded sections
for the nucleic acid amplification from individual cells. 25A.2 includes basic protocols for the preparation of single
cells from solid tissues for analysis by PCR.
[3] Wittwer et al. 1997: Continous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22: 130–138.
[4] Going and Lamb 1996: Practical histological microdissection for PCR analysis. Journal of Pathology 179: 121–124.
[5] Moskaluk and Kern 1997: Microdissection and polymerase chain reaction amplification of genomic DNA from
histological tissue sections. American Journal of Pathology, 150: 1547 – 1552.
[6] Fin et al. 2000: Immunostaining for cell picking an real-time mRNA quantitation.Amercian Journal of Pathology,
157: 1459–1466.
[7] Fend et al. 1999: Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.
American Journal of Pathology, 154: 61 – 66.
Tips for Piezo Power Microdissection (PPMD)
Micropipette with Filtertip MDS
The Filtertip MDS is designed to be
used only once for the transfer of
liquids (e.g. liquids containing dissected
cell material). It is therefore
not intended for use with other commercial
pipettes.
Cutting tool with MicroChisel
The MicroChisel can be autoclaved.
It can also be wiped or rinsed with
common cleaning agents (e.g.
agents used to remove DNA or
RNases).
The stainless steel MicroChisel is
extremely sharp and can be used
one or more times, depending upon
the application at hand. The actual
tip (1 µm at its tapered end) is set
in a 1 mm steel tube.
The Filtertip MDS, with an inner tip
diameter of 150 µm, has a filling volume
of approx. 30 µl. It can be autoclaved.
If it is left standing in organic
liquids for prolonged periods, this
may result in changes to the surface
and in the aspiration behavior.
PPMD step by step
Mounting the tools
The cutting tool (MicroChisel) and
the aspirating tool (Filtertip MDS)
can be mounted on the right or the
left-hand side of the microscope,
depending on the users preferences.
Mount the cutting tool with the Micro-
Chisel at a steep angle of approx. 40
– 45°.
According to experiences with several
tissues, we recommend to mount the
aspiration side with Filtertip MDS at an
angle of approx. 40°. The angle can be
varied according to the tissue type and
properties of the liquid, you are using.
Cutting side
1. Focus on the MicroChisel.
Choose the magnification according
to the tissue you are working on.
2. Set the parameters.
The frequency is set to 25 – 55 kHz,
in correspondence with the interaction
between the MicroChisel and
the tissue (operating manual page 14).
Set the amplitude to approx. 50 %.
3. The MicroChisel is activated by
pressing the appropriate e.g. right
foot control. What should be seen:
●The orange LED marked as
“External control” lights up.
●Under the microscope you will see
the MicroChisel oscillating and then
you fine tune the frequency and amplitude,
by observing the interaction
of the MicroChisel with the tissue.
●Irregular or very strong swinging
of the MicroChisel should be
avoided.
●Setting a Z-limit by pressing the
"Limit" button (TransferMan NK 2)
is very practical for the protection
of the tissue.
The tissue fragments now detaches
from the glass slide and into subcellular
particles.
Fig. 6 illustrates microdissection of cell
areas from human colon mucosa.
Aspiration side
1. Bring the Filtertip MDS into focus.
Choose the magnification according
to the tissue you are working on.
When starting the Filtertip is placed
approx. 10 - 20 µm apart from the
MicroChisel.
2. Set the parameters.
In this application - as we want to
gain our material by aspirating very
small particles – we are going to use
the continuous uptake mode
(Uptake Cont), which can be triggered
by the second foot control or
alternatively by pressing the appropriate
keys of the hand control. Set
the speed at the control board to > 5.
● Preadjust everything with a
control slide.
● Set the "Dispensed volume" of
the pipette to approx. 80 %
before you start. Otherwise
capillary forces might trick you.
Microdissection of cells and
collecting the material
● Add 15 μl xylene onto the glass
slide, bring area of interest below
the MicroChisel and lower
MicroChisel to working limit.
● Cut areas of interest while the
pipette is in HOME- Postion.
● Lower pipette immediately after
cutting. Touch the glass slide with
the MicroChisel switched on. The
ultrasonic effect resuspends the
remaining particles. Press the
foot or hand control to start aspirating
in continuous mode.
Collect as much of the xylene
containing the sample as possible.
● Press the HOME function of your
Eppendorf Manipulator to raise
the aspirating tool and transfer
xylene containing generated tissue
particles to a 0.5 ml tube by
pressing "Disp" or the right control
button.
● Add another 15 µl of xylene and
repeat the last two steps.
You might like to repeat this for the
following reasons:
● To optimize the amount of sample
collected.
● For cleaning the slide, if different
areas of a heterogeneous tissue
should be collected in the next
step.
Keep the cut material with xylene on
ice. Please continue to process the
material not later than 0.5 to 1 h
after collection in order to optimize
your yield.
Total RNA of the cell clusters and
cell particles microdissected were
extracted with RNA isolation kits [1].
Evaporate the xylene of your sample
in a vacuum concentrator (Eppendorf
Concentrator 5301) immediately
before you start with the downstream
applications.
Remarks and troubleshooting
It is not possible to aspirate the particles,
as they stick to the glass slide.
Do the following:
● Touch the glass slide with the
MicroChisel switched on. The
ultrasonic effect resuspends the
particles.
Although the MicroChisel oscillates
well, the cutting outline is not correct.
● The angle of mounting of the cutting
tool may not be steep
enough (approx. 40 - 45°).
● The MicroChisel has to be replaced;
it might be blunt.
The aspirating tool is not sucking
sufficiently.
● Please check the condition of the
black O-ring in the nose cone of
the adapter (operating manual
page 15). (You can do this with
the help of a thin metal tool,
which is available e.g. in the
Service Kit Order Number
5176 195.004).
The section tears because the
MicroChisel does not oscillate.
● The MicroChisel is pressing too
heavily on the object carrier and
does not oscillate freely. Adjust
the height using the micromanipulator.
● Change the frequency and/or the
amplitude to achieve better oscillation.
Not all particles are aspirated.
● Add approx. 15 to 20 µl of waterfree
solution to the tissue and
aspirate this volume in a Filtertip
MDS.
If you are using slides from an archive
with coverslips,
● you can leave the sample in
xylene overnight.
Using different cutting strategies
depending on the characteristics of
the tissue: Dry or wet?
● In this paper [1] Harsch et al. describe
a cutting technique in which
the relevant tissue is covered with
xylene while small particles are
cut away. This can be varied,
depending upon the particular tissue
you are working on.
● You can also cut dry if your tissue
separates into big pieces instead of
particles of dust. In this case:
a. cut the relevant tissue. An "island of tissue" is left behind.
b. cover that island with a few μl of xylene.
c. switch off the MicroChisel,
carefully remove the "island" or
single cell by scratching.
d. use the pipette in the stepwise
mode, activated by the left
key of the hand control (volume
and speed of the uptake must be
optimized for your tissue: for big
pieces use a relatively large volume
at low speed).
If you are interested in a very small
tissue area down to single cell level
● You can:
a. first clean the area around these cells with the MicroChisel (this should be done under a cover of xylene),
b. aspirate these particles,
c. discard Filtertip MDS, take a fresh Filtertip MDS and add xylene if necessary,
d. switch off the MicroChisel, carefully remove the "island" or single cell by scratching and
e. aspirate the "island".
Short protocol for PPMD™ from cryosections
● Preadjust everything with a control slide (including resonance, frequency and amplitude of MicroChisel and
dispensed volume to approx. 80 %)
● Check adjustment with the sample slide, including Z-limit, before adding xylene
● Use HOME for the aspiration side and CLEAN with 20 - 50 µm on the cutting side
● Add 15 µl xylene, position area of interest beneath the MicroChisel and lower MicroChisel to working limit
● Cut area of interest and lower pipette immediately afterwards
● Touch glass slide in area where the cells were removed to stir up all dissected cells and start aspiration of total
volume of xylene
● Use HOME on the aspiration side and transfer xylene with sample to a 0.5 ml tube by pressing DISPENSE (Disp)
● Add another 15 µl of xylene and repeat the last two steps
● OPTION: In some cases you may wish to repeat flushing the slide for dissected material
● Put collected sample on ice immediately and store at - 20 °C, if further processing is not possible shortly after
the dissection
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