Quality assurance of RNA derived from
laser microdissected tissue samples
obtained by the PALM® MicroBeam System
using the RNA 6000 Pico LabChip® kit.
|
|
Gene expression profiling of material isolated by microdissection has
become a very popular method for analyzing cellular behavior in a micro
scale and is used in a wide range of research and clinical applications.
Laser-assisted microdissection allows isolation of RNA from specific cell
populations out of their surrounding tissue environment. Usually, the
sample yield that can be obtained by this method is too low for quality
control of RNA using a standard technique. On the other hand, extraction
of high quality RNA is crucial for all subsequent steps and the success of
the overall experiment. Since the introduction of the Agilent RNA 6000
Pico LabChip® kit, it is possible for the first time to analyze total RNA
samples in concentrations down to 200 pg/µl, which is in the range of the
sample concentration one can expect from microdissected tissue or cells.
In this study we analyzed RNA samples that were obtained by laser
microdissection and pressure catapulting (LMPC; PALM® MicroBeam
System) from tissue sections. The influence of different staining procedures
and RNA extraction kits on RNA quality was investigated. RNA was
tested for quality and approximate yield, as well as the reproducibility
within homologous sample replicates. The data revealed clear differences
between the different isolation kits and also between the staining
procedures that were used.
|
The analysis of gene expression
has become one of the major
scientific tools for understanding
the behavior of cells in vivo and
in vitro and is used in many biological
and medical applications.
Using microarray hybridization,
the abundance of a large number
of transcripts can be analyzed in
a single experiment. Comparison
of different expression patterns
enables scientists to correlate the
changes in the transcriptome to
various external factors influencing
the cellular behavior. Especially in
clinical research there is the need
for methods that allow expression
analysis from very specific cells.
Prerequisite for meaningful results
are samples of high purity without
contamination from unwanted
cells that could potentially interfere
with detection of important
transcripts. Laser-assisted
microdissection with the
PALM®MicroBeam system allows
isolation of individual cells in a
simple and fast way. Using this
technology, researchers are able
to procure pure cell populations
from specific areas of tissue
sections for isolation of DNA,
RNA, and proteins with visual
control using a microscope. An
UV-A-laser-mediated process
dissects selected specimen from
various sources and transfers
them contact free directly into
collection vessels for subsequent
extraction (see figure 1). Together
with novel methods for linear
RNA amplification it is possible to
perform gene expression profiling
analyses even from very limited
amount of cell material. The most
|
|
critical parameter for the success
of such an experiment is the
integrity and purity of the RNA.
|
Experimental case study
|
Tissue preparation and microdissection
|
Snap-frozen mouse liver tissue
stored at –80 °C was cut in 7 µm
serial sections on a cryotome at
–25 °C. The sections were transferred
to PALM® MembraneSlides
(1mm glass slides covered with a
1.35 µm thin Polyethylene-naphthalate-
membrane to facilitate the
laser pressure catapulting procedure)
and air dried for 10 seconds.
After a fixation step of 5 minutes
in 70% ethanol at -20 °C and a
short wash in RNase free water
(10 seconds) the sections were
further processed according to
standard histochemical procedures.
Depending on the subsequent
tests either Hematoxylin/Eosin
(HE), Nuclear Fast Red (NFR),
Methylgreen (MG) or Methylene
Blue (MB) staining was applied.
After staining and a short increasing
ethanol series the sections
were air-dried and either used
immediately or refrozen at –20 °C
for up to 2 days. The PALM® Micro-
Beam System was used to precisely
excise the selected tissue areas
with the UV-A-laser and then to
catapult the areas of interest
contact free against gravity into
collecting caps filled with 12 µl
of RNase-free water. For lysis
the isolated material was then
immediately mixed by inversion
with the respective lysis buffers
for RNA extraction (see below).
|
RNA Purification
|
For this study RNA isolation kits
from 3 different manufacturers
were used. Two are based on a
column-purification, while the
third one uses an extraction-/precipitation-
based approach. RNA
cleanup was carried out according
to the manufacturers’ protocols
with the only variation that uniformly
a volume of 30 µl of elution
buffer or water was used in the
final step to collect the purified
RNA. For the column-procedures
the elution process was repeated
once with the primary eluate of
30 µl to improve the yield.
The scope of this work was the
demonstration of feasibility to perform
RNA sample quality control
from microdissected material and
not the evaluation of commercially
available RNA extraction kits.
Therefore, the manufacturers are
not explicitly named here, but
instead referred to as manufacturer
A, B, and C.
|
RT-PCR
|
Selected RNA samples were
reversely transcribed with the
First Strand cDNA Synthesis Kit
for RT PCR (AMV) cDNA Synthesis
Kit for RT-PCR (Roche)
according to the manufacturers’
protocol. Briefly, 5-8 µl of each
RNA solution were transcribed by
AMV-RTase with random primers
in a total volume of 20 µl for one
hour at 42 °C. For the subsequent
quantitative RT-PCR analyses
1-2 µl of each cDNA-solution
were used as templates. The PCR
amplification of the cDNA was
performed in a LightCycler® instrument (Roche) in 20 µl reactions
using protocols and components of
the QuantiTect SYBR Green PCR-Kit
(Qiagen). cDNA-specific primers
for murine PBGD (porphobilinogen
deaminase) were used as model
system producing a PCR-fragment
of 154 bp. Resulting crossing-point
values were used to compare
extraction efficiencies of different
kits or influences of different
staining procedures.
|
RNA 6000 Pico Assay Protocol
|
All chips were prepared according
to the instructions provided with
the RNA 6000 Pico LabChip Kit.
In brief, 550 µl of the RNA 6000 Pico
gel matrix were placed on a spin
filter, centrifuged at 1500 g and
divided in 65 µl aliquots. After
addition of 1µl RNA 6000 Pico dye
concentrate to one gel aliquot,
the mixture was vortexed and
centrifuged at 13000 g for 10 min.
A RNA 6000 Pico chip was filled
with gel-dye mix using the chip
priming station, followed by
addition of Conditioning Solution
and Marker. 1 µl of RNA 6000
ladder (Ambion) and RNA samples
were added in the designated wells,
the chip was vortexed for 1 minute
and run on an Agilent 2100 bioanalyzer.
|
Results and Discussion
|
RNA quality and yield obtained by different
isolation kits
|
All 3 kits tested in this study
appeared to work well together
with the RNA 6000 Pico assay
(figure 2). For the comparison
of the different kits we used a
standard HE tissue staining and
dissected areas corresponding to
1000, 2000 and 3000 cells. Quality
and yield of the isolated RNA
showed a high variability with
respect to the different kits and
also within the replicates of one
specific method. This may be
caused either by differences in the
tissue samples (different areas
were isolated from the same
section) or by some variances in
sample handling. On average the
extraction-/precipitation-based
method (manufacturer C) showed
a higher RNA recovery than the
two column-based methods,
which is most probably caused by
irreversible RNA-binding at the
column material. The average
yield (manufacturer C) obtained
from a 1000 cell area was approximately
700 pg/µl when resolving
the pellet in 30 µl of water. This
would result roughly in a theoretical
average RNA recovery rate of
approx 21 pg per cell. In contrast
to this the yield obtained by the
two column-based kits varied
between approx. 130 and 380 pg/µl
(1000 cells; 30 µl elution volume).
Note that the quantitative results
from an analysis with the RNA
6000 Pico kit are somewhat
dependent on the salt contentof a
sample. Only a rough estimation
of the RNA concentration of a
sample can be obtained.
|
|
RNA quality and yield obtained from
tissue stained with different methods
|
In this test, we compared RNA
quality and yield when using
different standard histochemical
staining methods. Therefore, we
dissected areas corresponding to
2000 cells and purified the RNA
using the kit of manufacturer C
(3 replicates for each sample).
Serial tissue sections were stained
using either Hematoxylin/Eosin
(HE), Nuclear Fast Red (NFR),
Methylgreen (MG) or Methylene
Blue (MB). In general, all four
staining procedures were compatible
with the RNA Pico Assay, but
significant differences in RNA
yield and quality were observed.
Best results were obtained using
the MG stain (average yield:
approx. 3100 pg; highly intact
RNA; see fig. 3A). HE (figure 3B)
and NFR staining (figure 3C) gave
very similar results in terms of
RNA yield and quality (average
yield: approx. 1600 pg; slightly
degraded RNA). In the case of MB
staining, ambiguous results were
observed (average yield: approx.
730 pg; partially degraded RNA;
figure 3D). The 28 S band is
almost entirely missing, which
could be due to RNA degradation
or due to a salt content that
exceeds the salt specifications of
the Pico kit. RNA-extraction with
the kit of manufacturer B from
the same sections with all four
staining methods yielded slightly
different relations (see in RT-PCR
results below), and overall lower
concentration levels as expected
from the comparison of the kits
from above. It should be noted
that this short investigation does
not put a value on the use of
certain cell stains for work with
microdissected material but rather
points out the value of the RNA
6000 Pico kit for optimization of
experimental protocols.
|
|
Correlation of the results obtained
by the RNA Pico Assay to real-time
RT-PCR
|
Comparison of RNA extraction kits:
|
All 3 sample extraction kits that
were tested yielded good quality
RNA that could be used for
RT-PCR (figure 4). For the comparison
we used HE-stained sections
to microdissect and catapult
areas of 1000 cells each (3 replicates
per sample). After RNA
isolation with the three different
kits real-time RT-PCR with the
LightCycler‚ was performed. The
amount of cDNA used as template
was half of the RNA used for the
2100 bioanalyzer analysis (fig. 4A).
The specificity of the PCR-fragments
was proven by the melting
curve (figure 4B). The crossing
points (Cp)* of the growth curves
(figure 4C) being a scale for the
initial specific RNA-amount were
close together (about one cycle
distance each) correlating to the
Bioanalyzer values although
Manufacturer A was overestimated
there probably due to the
DNA-contamination (figure.1).
|
|
Comparison of staining
procedures
|
In this test serial tissue
sections were stained using
either Hematoxylin/Eosin
(HE), Nuclear Fast Red (NFR),
Methylgreen (MG) or Methylene
Blue (MB). Areas corresponding
to 2000 cells were dissected,
the RNA purified using Manufacturer
B (3 replicates each)
and analyzed with the 2100
bioanalyzer (figure 5A) and
the Light-Cycler® as above.
The cDNA input for the PCR
corresponded to a fourth of
the RNA amount used for the
2100 bioanalyzer run. The melting
curve proves the specificity
of the products (figure 5B),
while the growth curve reflects
the starting amount of RNA
(figure 5C). In contrast to the
test with Manufacturer C (see
above) NFR was the optimal
stain, though the Cp-values lay
very close together (only half
a cycle difference) except
for MB which showed worse
results. (CpNFR: 34,7; CpHE:
35,2; CpMG: 35,8; CpMB: 37,6).
This real-time RT-PCR results
again correlated well with the
2100 bioanalyzer values.
|
|
Conclusion
|
RNA derived from laser-microdissected
tissue isolated by the
PALM®MicroBeam system is of
high quality and can be analyzed
efficiently using the RNA 6000
Pico assay with the Agilent 2100
bioanalyzer. RNA-purification kits
of different manufacturers and
various common staining procedures
have been tested and were
compatible with the Pico Assay.
Nevertheless, it seems to be advisable
that any extraction protocol
and staining method should be
tested in combination to find
the optimal procedure for tissue
microdissection experiments.
The RNA 6000 Pico kit is well
suited to show differences in RNA
quality and, therefore, is an ideal
tool to optimize experimental
conditions. Its unprecedented
sensitivity allows for the first
time quality control in the context
of microdissection experiments
ensuring successful gene expression
profiling experiments.
|