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High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells

  • September 2014
  • Journal of Biotechnology 192(4)
DOI:10.1016/j.jbiotec.2014.09.016
Authors:
Helén Nilsson
Helén Nilsson
Helén Nilsson
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Krzysztof Krawczyk at Skåne University Hospital
Krzysztof Krawczyk
Martin E Johansson at University of Gothenburg
Martin E Johansson
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Abstract and Figures

Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.
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High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cel
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Journal
of
Biotechnology
192
(2014)
62–65
Contents
lists
available
at
ScienceDirect
Journal
of
Biotechnology
j
ourna
l
ho
me
pa
ge:
www.elsevier.com/locate/jbiotec
Short
communication
High
salt
buffer
improves
integrity
of
RNA
after
fluorescence-activated
cell
sorting
of
intracellular
labeled
cells
Helén
Nilsson∗,1,
Krzysztof
M.
Krawczyk1,
Martin
E.
Johansson.
Department
of
Laboratory
Medicine,
Center
for
Molecular
Pathology,
Lund
University,
Sweden
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
17
June
2014
Received
in
revised
form
10
September
2014
Accepted
19
September
2014
Available
online
30
September
2014
Keywords:
RNA
integrity
Intracellular
antibody
labeling
Fluorescence-activated
cell
sorting
Fixation
NaCl
a
b
s
t
r
a
c
t
Over
the
past
years,
massive
progress
has
been
made
in
the
ability
to
collect
large-scale
gene
expres-
sion
data
from
a
limited
sample
size.
Combined
with
improvements
in
multiplex
flow
cytometry-based
techniques,
this
has
made
it
possible
to
isolate
and
characterize
specific
cellular
subtypes
within
heteroge-
neous
populations,
with
a
great
impact
on
our
understanding
of
different
biological
processes.
However,
sorting
based
on
intracellular
markers
requires
fixation
and
permeabilization
of
samples,
and
very
often
the
integrity
of
RNA
molecules
is
compromised
during
this
process.
Many
attempts
have
been
made
to
improve
the
quality
of
nucleic
acids
from
such
samples,
but
RNA
degradation
still
remains
a
limiting
factor
for
downstream
analyses.
Here
we
present
a
method
to
isolate
high
quality
RNA
from
cells
that
have
been
fixed,
permeabilized,
intracellularly
labeled
and
sorted.
By
performing
all
incubation
steps
in
the
presence
of
a
high
salt
buffer,
RNA
degradation
was
avoided
and
samples
with
remarkable
integrity
were
obtained.
This
procedure
offers
a
straightforward
and
very
affordable
technique
to
retrieve
high
quality
RNA
from
isolated
cell
populations,
which
increases
the
possibilities
to
characterize
gene
expres-
sion
profiles
of
subpopulations
from
mixed
samples,
a
technique
with
implications
in
a
broad
range
of
research
fields.
©
2014
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
1.
Introduction
Awareness
regarding
the
importance
of
cellular
heterogeneity
in
complex
tissues
and
samples
is
increasing,
and
much
knowledge
would
be
gained
would
it
be
possible
to
obtain
high
quality
RNA
from
cells
isolated
from
heterogeneous
populations,
for
example
from
primary
tissue.
Flow
cytometry
based
techniques
are
pow-
erful
tools
for
isolation
of
cells,
but
depend
heavily
on
labeling
of
extracellularly
facing
antigens
for
retrieval
of
intact
RNA.
Intracel-
lular
labeling
of
fixed
and
permeabilized
cells
allows
for
separation
of
cell
populations
using
fluorescence-activated
cell
sorting
(FACS).
The
necessary
fixation
and
permeabilization
steps
before
intra-
cellular
antibody
staining
however
has
profound
effects
on
RNA
integrity
(Russell
et
al.,
2013).
The
single
stranded
nature
of
RNA
molecules
renders
them
sensitive
to
degradation,
and
crosslink-
ing
resulting
from
fixation
has
well
known
detrimental
effects
on
RNA
integrity
and
often
precludes
downstream
analyzes
such
as
∗Corresponding
author
at:
Center
for
Molecular
Pathology,
Lund
University,
Department
of
Laboratory
Medicine
Malmö,
Skåne
University
Hospital,
Jan
Walden-
ströms
gata
59,
20502
Malmö,
Sweden.
Tel.:
+46
040336269;
fax:
+46
040337063.
E-mail
address:
helen.nilsson@med.lu.se
(H.
Nilsson).
1These
authors
contributed
equally
to
this
paper.
gene
arrays
and
quantitative
real-time
PCR
(QPCR)
(Auer
et
al.,
2003;
Fleige
and
Pfaffl,
2006).
Several
attempts
have
been
made
to
improve
protocols
in
order
to
isolate
RNA
from
sorted
cells
with
acceptable
quality,
however,
RNA
integrity
values
remain
problem-
atically
low
(Diez
et
al.,
1999;
Nishimoto
et
al.,
2007;
Iglesias-Ussel
et
al.,
2013).
Different
RNA-preserving
fixation
solutions
has
also
been
developed,
but
these
are
often
costly
and
not
compatible
with
antibody
labeling
(Zaitoun
et
al.,
2010).
Here
we
present
a
method
to
obtain
high
quality
RNA
from
intracellularly
labeled
and
sorted
cells
in
a
straightforward
and
affordable
way.
We
show
that
the
presence
of
a
high-salt
buffer
protects
RNA
from
degradation
during
antibody
staining
and
sor-
ting
steps,
which
enables
the
collection
of
high
quality
RNA
from
cells
sorted
based
on
intracellular
markers.
2.
Materials
and
methods
2.1.
Cell
culture
786-O
and
Jurkat
cells
were
cultured
in
DMEM
high
glucose
(Thermo
scientific)
supplemented
with
10%
fetal
bovine
serum
and
1%
Penicillin-Streptomycin
solution
(Thermo
scientific)
at
37 ◦C
and
5%
CO2.
http://dx.doi.org/10.1016/j.jbiotec.2014.09.016
0168-1656/©
2014
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
H.
Nilsson
et
al.
/
Journal
of
Biotechnology
192
(2014)
62–65
63
Fig.
1.
(A)
Electropherograms
from
Bioanalyzer
showing
the
poor
integrity
of
RNA
isolated
from
cells
after
fixation,
permeabilization,
antibody
labeling
and
sorting.
Fixation
and
permeabilization
alone
(B),
as
well
as
fixation
and
permeabilization
followed
by
sorting
(C)
results
in
RNA
of
high
quality.
(D)
Degraded
RNA
from
cells
isolated
after
fixation,
permeabilization,
and
antibody
labeling.
2.2.
Fixation
and
permeabilization
At
90%
confluency
cells
were
trypsinized
and
counted.
Cells
were
fixed
in
4%
paraformaldehyde
solution
(PFA)
for
10
min
at
room
temperature.
Fixed
cells
were
kept
on
ice
for
1
min
before
permeabilization
by
addition
of
ice-cold
100%
methanol
to
a
final
concentration
of
90%.
Cells
were
subsequently
incubated
on
ice
for
30
min
and
stored
at
−20 ◦C.
2.3.
Antibody
staining
Fixed
and
permeabilized
cells
kept
in
methanol
were
cen-
trifuged
for
5
min
at
1000
×
g
at
4◦C.
Supernatant
was
removed
and
cells
were
washed
in
PBS
or
2
M
NaCl
in
PBS
to
remove
the
remaining
methanol.
Cells
were
incubated
in
blocking
buffer
(0.5%
BSA
in
PBS,
with
or
without
2
M
NaCl
for
10
min
in
room
tempera-
ture.
Staining
with
primary
conjugated
anti-human
cytokeratin
7/8
antibody
(Alexa
Fluor
647,
clone
CAM5.2,
BD
Biosciences)
was
per-
formed
for
20
min
at
room
temperature
in
blocking
buffer.
Excess
antibody
was
washed
away
using
blocking
buffer.
Antibody
inten-
sity
was
analyzed
on
a
FACS
Calibur
(BD
Biosciences).
2.4.
Fluorescence
activated
cell
sorting
For
cell
sorting
a
FACSAria
Cell
Sorter
(BD
Biosciences)
was
used.
As
a
precaution
to
avoid
RNA
degradation
cells
were
collected
in
4
M
NaCl
and
1%
BSA
in
PBS.
2.5.
RNA
isolation
and
quality
control
RNA
was
isolated
using
Qiagen’s
FFPE
kit
according
to
the
man-
ufacturer’s
protocol.
For
all
samples
the
highest
recommended
volume
of
PKD
buffer
(240
␮L)
was
used
to
avoid
interference
from
remnants
of
high
salt
buffer.
RNA
integrity
was
assessed
with
Agi-
lent
RNA
6000
Nano
kit
and
Agilent
2100
Bioanalyzer
(Agilent
Technologies).
2.6.
cDNA
synthesis
and
quantitative
real-time
PCR
cDNA
synthesis
was
performed
using
MultiScribe
Reverse
Transcriptase
enzyme
and
random
hexamers
(Applied
Biosystems).
Quantitative
real-time
PCR
was
performed
with
SYBR
Green
PCR
Master
Mix
on
a
7300
Real-Time
PCR
System
(Applied
Biosystems).
The
comparative
Ct
method
was
used
to
quantify
relative
RNA
levels
and
three
separate
housekeeping
genes
(HMBS,
RPL13A,
YWHAZ)
were
used
for
normalization.
Primer
sequences
used
were
CK8
forward:
5-AGGGCTGACCGACGAGAT-3,
reverse:
5-
CACCACAGATGTGCTCGAGA-3,
HMBS
forward:
5-GGCAATGCGG-
CTGCAA-3,
reverse:
5-GGGTACCCACGCGAATCAC-3,
RPL13A
forward:
5-CCTGGAGGAGAAGAGGAAAGAGA-3,
reverse:
5-
TTGAGGACCTCTGTGTATTTGTCAA-3,
YWHAZ
forward:
5-ACTTTT-
GGTACATTGTGGCTTCAA-3,
reverse:
5-CCGCCAGGACAAA-
CCAGTAT-3.
3.
Results
and
discussion
3.1.
RNA
integrity
is
lost
during
antibody
incubation
of
fixed
and
permeabilized
cells
The
detrimental
effects
on
RNA
integrity
caused
by
formalin
crosslinking
and
antibody
incubation
steps
have
made
it
difficult
to
obtain
RNA
of
adequate
quality
from
complex
samples
where
fixation
and
intracellular
labeling
is
required
before
cell
sorting.
To
illustrate
this
problem,
we
performed
the
procedure
in
the
human
renal
carcinoma
cell
line
786-O.
Cells
were
fixed
in
4%
paraformaldehyde,
permeabilized
using
methanol
and
labeled
with
a
primary
Alexa
647
conjugated
cytokeratin
7/8
antibody
(CK7/8),
that
should
result
in
intracellular
staining
of
these
cells.
Positive
cells
were
collected
using
FACS
and
RNA
was
isolated.
The
qual-
ity
of
the
obtained
RNA
was
determined
on
a
Bioanalyzer.
The
RNA
integrity
number,
or
RIN
value,
obtained
from
the
Bioanalyzer
gives
a
measurement
of
the
degree
of
degradation
that
has
occurred,
where
a
RIN
value
of
10
corresponds
to
intact
RNA
(Schroeder
et
al.,
64
H.
Nilsson
et
al.
/
Journal
of
Biotechnology
192
(2014)
62–65
2006).
As
shown
in
Fig.
1A,
the
process
of
fixation,
permeabiliza-
tion,
antibody
labeling
and
sorting
resulted
in
RNA
with
a
RIN
value
as
low
as
2.1.
We
next
evaluated
the
effect
of
each
of
these
steps
on
RNA
integrity.
Fixation
and
permeabilization
alone
had
no
negative
effect
(Fig.
1B);
also
sorting
of
fixed
and
permeabilized
cells
could
be
performed
without
compromising
RNA
quality
(Fig.
1C).
How-
ever,
RIN-values
of
RNA
from
cells
that
were
fixed,
permeabilized
and
incubated
with
antibody
were
considerably
lower
(Fig.
1D),
indicating
that
the
blocking
and
antibody
incubation
steps
had
a
detrimental
effect
on
RNA
integrity.
3.2.
Incubation
in
high
salt
buffer
preserves
RNA
integrity
It
has
previously
been
reported
that
antibody
incubation
in
a
high
salt
buffer
improves
the
quality
of
RNA
obtained
after
laser
micro-dissection
of
specifically
stained
cell
populations
(Brown
and
Smith,
2009).
This
method
yielded
RNA
of
better
quality
than
the
commercially
available
RNA
preserving
agent
RNAlater,
and
enabled
prolonged
antibody
incubation
times
without
compro-
mising
RNA
integrity.
We
therefore
set
out
to
test
whether
this
method
could
also
be
applicable
on
cells
isolated
by
FACS.
In
order
to
test
this
method,
we
performed
blocking
and
antibody
labeling
in
the
presence
of
2
M
NaCl.
RIN-values
of
RNA
iso-
lated
from
cells
incubated
under
these
conditions
were
markedly
improved,
as
illustrated
in
Fig.
2A.
Also
when
the
cells
were
sorted
after
fixation,
permeabilization,
blocking
and
antibody
labeling
at
room
temperature
in
high
salt
buffer,
RNA
with
RIN
values
as
high
as
7.9
were
obtained
(Fig.
2B).
These
results
indicate
that
the
high
salt
buffer
protects
RNA
from
fragmentation,
result-
ing
in
distinctly
improved
RNA
quality.
No
difference
in
total
RNA
yield
was
observed
whether
or
not
the
high
salt
buffer
was
used.
3.3.
High
salt
buffer
do
not
have
major
impact
on
antibody
binding
efficiency
Antibody
binding
to
specific
epitopes
can
be
sensitive
to
changes
induced
by
fixation
and
other
procedures.
To
assure
that
the
high
salt
buffer
did
not
compromise
the
specificity
or
affinity
of
the
anti-
body,
786-O
cells
were
stained
with
the
CK7/8
antibody
in
PBS
or
high
salt
buffer,
and
the
intensity
of
the
staining
was
analyzed
by
Fig.
2.
Antibody
incubation
of
fixed
and
permeabilized
cells
performed
in
the
pres-
ence
of
high
salt
buffer
results
in
RNA
of
high
quality
(A),
also
when
cells
are
sorted
after
labeling
(B).
flow
cytometry.
786-O
cells
stained
in
PBS
buffer
were
positive
for
CK7/8,
as
expected.
Antibody
incubation
in
high
salt
buffer
resulted
in
a
slight
decrease
in
staining
intensity,
but
the
same
proportion
of
cells
were
positive,
and
the
signal
was
still
well
separated
from
the
signal
of
unstained
cells
(Fig.
3A
and
B).
In
contrast
to
786-O
cells,
human
T
lymphocyte
Jurkat
cells
do
not
express
CK7/8,
and
should
thus
be
negative
for
this
antibody.
As
expected,
all
Jurkat
cells
were
negative
for
this
antibody
regardless
of
whether
the
antibody
incubation
occurred
in
PBS
or
high
salt
buffer,
indicating
that
high
salt
buffer
did
not
induce
unspecific
staining
(data
not
shown).
Fig.
3.
Flow
cytometry
results
showing
the
intensity
of
CK7/8
Alexa
647
antibody
staining
of
positive
(red)
786-O
cells,
where
antibody
incubation
was
performed
in
PBS
(A)
or
high
salt
buffer
(B),
compared
to
unstained
cells
(blue).
(For
interpretation
of
the
references
to
color
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
the
article.)
H.
Nilsson
et
al.
/
Journal
of
Biotechnology
192
(2014)
62–65
65
Fig.
4.
Electropherograms
of
RNA
isolated
from
a
mixture
of
786-O
and
Jurkat
cells
after
fixation,
permeabilization
and
CK7/8-antibody
labeling
followed
by
sorting
based
on
CK7/8
positivity.
Both
CK7/8-positive
(A)
and
negative
(B)
populations
have
high
RIN
values.
(C)
QPCR
results
showing
expression
of
CK8
in
the
population
that
stained
positive
for
this
antibody,
while
negative
cells
express
very
low
levels
of
this
marker.
3.4.
High
salt
buffer
enables
preparation
of
high
quality
RNA
from
a
specific
cell
population
identified
and
collected
by
FACS
after
intracellular
antibody
labeling
To
illustrate
possible
applications
of
this
improved
method,
we
prepared
a
mixture
of
the
two
different
cell
types,
786-O
and
Jurkat
cells.
Cells
were
fixed,
permeabilized
and
stained
with
the
CK7/8
antibody
in
the
presence
of
high
salt
buffer
as
described
above.
Using
FACS,
positive
(786-O)
and
negative
(Jurkat)
cells
were
sepa-
rated
and
RNA
was
isolated
from
both
populations
(Fig.
4A
and
B).
Expression
of
cytokeratin
8
was
analyzed
in
these
samples
using
QPCR,
confirming
the
separation
of
the
two
populations
(Fig.
4C).
This
experiment
also
validates
that
the
quality
of
the
isolated
RNA
is
sufficient
for
cDNA
synthesis
and
QPCR
reactions.
An
additional
advantage
using
this
protocol
is
the
possibility
to
store
samples
before
analysis
by
FACS,
enabling
collection
of
large
datasets
that
can
be
analyzed
in
parallel.
It
also
allows
for
samples
to
be
shipped
to
other
locations
for
analysis,
something
that
can
be
useful
in
cases
of
multi-centric
collaborations
or
when
the
FACS
analysis
is
performed
at
distant
core
facilities.
4.
Conclusion
This
improved
method
opens
up
for
low-cost
FACS
based
cell
sorting
of
different
subtypes
of
cells
from
complex
populations,
using
intracellular
labeling
of
cells.
The
protocol
results
in
high
quality
RNA
suitable
for
further
downstream
characterization
by
a
broad
repertoire
of
molecular
biology
platforms.
Acknowledgments
The
authors
would
like
to
thank
Per
Anders
Bertilsson
at
Lund
University
Flow
Cytometry
Facility
for
excellent
technical
assis-
tance.
This
work
was
funded
by
Marianne
&
Marcus
Wallenberg
Foundation
(MMW2011.0078),
the
National
Association
against
Kidney
Disease
(NF130909),
Governmental
funding
of
Clinical
Research
within
the
National
Health
Service
(ALF)
(M2011/1816),
SUS
Foundations
and
Donations
(314650),
and
the
Malmö
General
Hospital
Research
Fund
(ASM131203MJ)
for
cancer
research.
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... Several studies have attempted to minimize the impact of fixation and permeabilization on RNA quality, with variable results (8)(9)(10)(11)(12)(13)(14)(15)(16). While two recent methods to isolate high-quality RNA from formaldehyde-fixed, saponin-permeabilized, FACsorted cell populations have shown promise, each method has potential limitations. ...
... Additionally, RNase inhibitors may only bind specific RNases, and this binding is typically reversible and highly dependent on temperature. Alternatively, the method proposed by Nilsson et al. utilizes a cost-effective high salt buffer (i.e., 2M NaCl) to broadly inactivate RNases (16). While this method is also effective in limiting RNA degradation, the use of a high-ionic buffer can alter antibody tertiary structure and may significantly reduce or alter antibody binding. ...
... The isolation and subsequent characterization of many cell types of interest require quantification of intracellular targets, including but not limited to cytokines, kinases, and transcription factors. Several prior publications have proposed methods for the isolation of high-quality RNA from fixed and permeabilized samples, including the use of RNase inhibitors, high-salt buffers, zinc-based buffers, as well as more modern, completely reversible fixatives (e.g., DSP, SPDP) (8)(9)(10)(11)(12)(13)(14)(15)(16); however, each of these methods has potential drawbacks. Zinc salt-based fixatives, which do not result in crosslinking, have been shown to maintain RNA integrity when coupled with saponin-permeabilization and intracellular staining, and their effect on downstream sequencing protocols remains unclear (10,11,77). ...
Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells
Article
Full-text available
  • Sep 2020
Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.
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... Samples were taken early in the competence state at 5.5 h and at a later stage at 6.5 h in order to gain insight into the progression of competence. Cells were preserved using 2 M sodium chloride to prevent degradation of RNA before FACS and sorted into 4 M NaCl due to dilution taking place during sorting (Brown and Smith, 2009;Nilsson et al., 2014). The suitability of NaCl as preserving agent for preventing RNA degradation in B. subtilis was confirmed by comparison with flash freezing in liquid nitrogen [Supporting Information S1(Sheet)A and (Sheet)B]. ...
... To prevent degradation of RNA, the cells were preserved with 2 M NaCl in PBS before FACS and sorted in to 4 M NaCl in PBS (Brown and Smith, 2009;Nilsson et al., 2014). The NaCl preservation method was tested by microarray analyses (Supporting Information S1A and B). ...
Analyses of competent and non‐competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence
Article
Full-text available
  • Apr 2020
Upon competence‐inducing nutrient‐limited conditions only part of the B. subtilis population becomes competent. Here, we separated the two sub‐populations by Fluorescence Assisted Cell Sorting (FACS). Using RNA‐seq we confirmed the previously described ComK regulon. We also found for the first time significantly down‐regulated genes in the competent sub‐population. The down‐regulated genes are not under direct control by ComK, but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell‐division and replication are halted. By investigating the proteome during competence we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post‐transcriptional level. In the competent sub‐population yhfW was newly identified as being highly up‐regulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK‐binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA‐seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC. This article is protected by copyright. All rights reserved.
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... Following myelin removal, the cells were fixed in 4% paraformaldehyde (Sigma Aldrich, 441244) and the nuclei were stained with 1 μg/ml Hoechst (Sigma Aldrich). The RNA was protected with high salt buffers as described previously (Jager et al., 2018;Nilsson, Krawczyk, & Johansson., 2014) and the SGCs were stained with anti-Glutamine synthetase antibody (Abcam, Ab49873, RRID:AB_880241). ...
... RNA was purified with the RNeasy FFPE Kit (Qiagen, Hilden, Germany, 73504) according to the manufacturer's instructions and as described previously (Jager et al., 2018;Nilsson et al., 2014). The F I G U R E 1 Isolation of SGCs. ...
Changes in the transcriptional fingerprint of satellite glial cells following peripheral nerve injury
Article
  • Feb 2020
Satellite glial cells (SGCs) are homeostatic cells enveloping the somata of peripheral sensory and autonomic neurons. A wide variety of neuronal stressors trigger activation of SGCs, contributing to, for example, neuropathic pain through modulation of neuronal activity. However, compared to neurons and other glial cells of the nervous system, SGCs have received modest scientific attention and very little is known about SGC biology, possibly due to the experimental challenges associated with studying them in vivo and in vitro. Utilizing a recently developed method to obtain SGC RNA from dorsal root ganglia (DRG), we took a systematic approach to characterize the SGC transcriptional fingerprint by using next‐generation sequencing and, for the first time, obtain an overview of the SGC injury response. Our RNA sequencing data are easily accessible in supporting information in Excel format. They reveal that SGCs are enriched in genes related to the immune system and cell‐to‐cell communication. Analysis of SGC transcriptional changes in a nerve injury‐paradigm reveal a differential response at 3 days versus 14 days postinjury, suggesting dynamic modulation of SGC function over time. Significant downregulation of several genes linked to cholesterol synthesis was observed at both time points. In contrast, regulation of gene clusters linked to the immune system (MHC protein complex and leukocyte migration) was mainly observed after 14 days. Finally, we demonstrate that, after nerve injury, macrophages are in closer physical proximity to both small and large DRG neurons, and that previously reported injury‐induced proliferation of SGCs may, in fact, be proliferating macrophages. We present the transcriptional fingerprint of SGCs from naïve and nerve‐injured mice. RNAseq 3 and 14 days after injury show initial regulation of cholesterol metabolism and subsequent immune regulation. Previously reported injury‐induced proliferation of SGCs are likely proliferating macrophages.
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... The instrument was calibrated using Cytometer Setup and Tracking beads (BD Biosciences). To maintain the integrity of the RNA in PFA-fixed cells, blocking and antibody labeling was performed in the presence of high salt buffer (2 M NaCl) 55 . ...
Fibrocytes boost tumor-supportive phenotypic switches in the lung cancer niche via the endothelin system
Article
Full-text available
  • Oct 2022
Fibrocytes are bone marrow–derived monocytic cells implicated in wound healing. Here, we identify their role in lung cancer progression/ metastasis. Selective manipulation of fibrocytes in mouse lung tumor models documents the central role of fibrocytes in boosting niche features and enhancing metastasis. Importantly, lung cancer patients show increased number of circulating fibrocytes and marked fibrocyte accumulation in the cancer niche. Using double and triple co-culture systems with human lung cancer cells, fibrocytes, macrophages and endothelial cells, we substantiate the central features of cancer-supporting niche: enhanced cancer cell proliferation and migration, macrophage activation, augmented endothelial cell sprouting and fibrocyte maturation. Upregulation of endothelin and its receptors are noted, and dual endothelin receptor blockade suppresses all cancer-supportive phenotypic alterations via acting on fibrocyte interaction with the cancer niche. We thus provide evidence for a crucial role of fibrocytes in lung cancer progression and metastasis, suggesting targets for treatment strategies.
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... 32 Careful selection of buffer can improve sorting outcomes such as the integrity of RNA post sorting. 33,34 The optimization of the protocol to be used for cell sorting is a lengthy and technical procedure that involves many variables includ- 45 ...
Applications of flow cytometry sorting in the pharmaceutical industry: A review
Article
Full-text available
  • Jan 2021
The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inocu-lum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures. K E Y W O R D S droplet cell sorting, flow cytometry sorting, mutant screening, pharmaceuticals, rare cell isolation, surface display 1 | INTRODUCTION This review deals with flow cytometry sorting, an analytical technique based on flow cytometry that enables cells to be sorted according to some cellular property. Flow cytometry is a sensitive and powerful tool that uses fluorescence and light scattering to simultaneously measure the physical and chemical properties of cells suspended in a fluid. Cytometry is a high throughput process that characterizes individual cells at very high rates and so can detect rare events in real time. Several cellular parameters can be measured simultaneously depending on the number of detectors available. The determination of these parameters is based on a wide range of fluorescent dyes and the ability to conjugate antibodies to fluorophores. Flow cytometry can characterize gene expression, 1-3 cell viability, 4-6 target binding affinity, 7,8 cell surface markers, 9-11 location of the cell in the cell cycle 12,13 and oxidative stress. 14,15 The main disadvantages of flow cytometry are that many dyes exhibit similar emission spectra 16 and that RNA integrity and cell viability may be compromised by the toxicity of these dyes. 17,18 Cell samples are usually subjected to a pre-treatment which can include centrifugation, tissue dissociation, and/or resuspension in a buffer. The suspended cell samples are then stained with fluorescent conjugated antibodies and/or dye(s) and introduced into the flow cytometer. Flow cytometers typically contains two fluid traveling in the same direction toward a flow cell: one containing the cell suspension and the other containing sheath fluid. Hydrodynamic focusing occurs as the slower stream, in this case containing the cell suspension , is confined within the faster flowing stream and a laminar flow profile is created. Ideally, the particles in the cell suspension line up in single file to be characterized one at a time by focusing a laser beam on each passing cell, measuring the energy re-emitted as fluorescence by the excited fluorophores to detectors located downstream. The detectors are most commonly photomultiplier tubes (PMT) which convert the emitted or scattered light into amplified electrical pulses that are processed by appropriate electronics to extract information like pulse height, area, length, and time. Forward and side scattering are related to particle's size and complexity (granularity) of the cell membrane, respectively. The emitted light is related to fluores-cence usually from antibodies conjugated to fluorophores, the occurrence of naturally fluorescent molecules or externally added fluorescent dyes. The fluorophores may be located intracellularly or on the cell membrane. Multiple fluorophores may be monitored
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... This alteration in the protocol preserved the quality of ICCS, and thus the distinction of different cytokine producers (5). To free RNA from protein complexes, we included a proteinase K digestion step prior to RNA isolation (12) (Fig. 1A). These adjustments yielded mRNA levels from fixed T cells comparable to "fresh" T cells that were prepared without permeabilization and formaldehyde fixation (SI Appendix, Fig. S1A). ...
CD29 identifies IFN-γ–producing human CD8 + T cells with an increased cytotoxic potential
Article
Full-text available
  • Mar 2020
Cytotoxic CD8 ⁺ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8 ⁺ T cells with a cytotoxic expression profile are lacking. Human CD8 ⁺ T cells can be divided into IFN-γ– and IL-2–producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8 ⁺ T cells revealed that IL-2 ⁺ cells produce helper cytokines, and that IFN-γ ⁺ cells produce cytotoxic molecules. IFN-γ ⁺ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29 ⁺ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.
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Different Resource Allocation in a Bacillus subtilis Population Displaying Bimodal Motility
Article
Full-text available
To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially. This is one reason why bimodal differentiation is primarily observed from the onset of the stationary phase when exponential growth ceases. An exception is the bimodal induction of motility in Bacillus subtilis , which occurs early during exponential growth. Several mechanisms have been put forward to explain this, including double negative-feedback regulation and the stability of the mRNA molecules involved. In this study, we used fluorescence-assisted cell sorting to compare the transcriptome of motile and non-motile cells and noted that expression of ribosomal genes is lower in motile cells. This was confirmed using an unstable GFP reporter fused to the strong ribosomal rpsD promoter. We propose that the reduction in ribosomal gene expression in motile cells is the result of a diversion of cellular resources to the synthesis of the chemotaxis and motility systems. In agreement, single-cell microscopic analysis showed that motile cells are slightly shorter than non-motile cells, an indication of slower growth. We speculate that this growth rate reduction can contribute to the bimodal induction of motility during exponential growth. IMPORTANCE To cope with sudden environmental changes, bacteria can use a bet-hedging strategy and generate different types of cells within a population, so called bimodal differentiation. For example, a Bacillus subtilis culture can contain both motile and non-motile cells. In this study we compared the gene expression between motile and non-motile cells. It appeared that motile cells express less ribosomes. To confirm this, we constructed a ribosomal promoter fusion that enabled us to measure expression of this promoter in individual cells. This reporter fusion confirmed our initial finding. The re-allocation of cellular resources from ribosome synthesis towards synthesis of the motility apparatus results in a reduction in growth. Interestingly, this growth reduction has been shown to stimulate bimodal differentiation.
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Applications of Flow Cytometry Sorting in the Pharmaceutical Industry
Article
  • Mar 2021
The paper reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: i‐ isolation of cell types, ii‐high throughput screening, iii‐ cell surface display, iv‐ droplet fluorescent‐activated cell sorting (FACS). Future opportunities are identified including: i‐ sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and ii‐ the use of population balance models in combination with FACS to design and optimize cell cultures.
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Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
Article
Full-text available
A simple method for extraction of high quality RNA from cells that have been fixed, stained and sorted by flow cytometry would allow routine transcriptome analysis of highly purified cell populations and single cells. However, formaldehyde fixation impairs RNA extraction and inhibits RNA amplification. Here we show that good quality RNA can be readily extracted from stained and sorted mammalian cells if formaldehyde is replaced by glyoxal—a well-characterised fixative that is widely compatible with immunofluorescent staining methods. Although both formaldehyde and glyoxal efficiently form protein-protein crosslinks, glyoxal does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. We find that RNA integrity is maintained through glyoxal fixation, permeabilisation with methanol or saponin, indirect immunofluorescent staining and flow sorting. RNA can then be extracted by standard methods and processed into RNA-seq libraries using commercial kits; mRNA abundances measured by poly(A)+ RNA-seq correlate well between freshly harvested cells and fixed, stained and sorted cells. We validate the applicability of this approach to flow cytometry by staining MCF-7 cells for the intracellular G2/M-specific antigen cyclin B1 (CCNB1), and show strong enrichment for G2/M-phase cells based on transcriptomic data. Switching to glyoxal fixation with RNA-compatible staining methods requires only minor adjustments of most existing staining and sorting protocols, and should facilitate routine transcriptomic analysis of sorted cells.
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Cellules Club et susceptibilité respiratoire environnementale
Thesis
  • Mar 2020
La BPCO (BronchoPneumopathie Chronique Obstructive) est une maladie chronique des voies aériennes où prédominent modifications structurelles et inflammation. Le but de ce travail a été d’étudier différents axes caractéristiques de cette pathologie comme l’hyperplasie des cellules à mucus, le déficit en cellules Club et la dérégulation des alarmines pour trouver de nouvelles cibles thérapeutiques.Tout d’abord, nous avons réalisé la carte d’identité des cellules Club issues de cultures en interface air-liquide (ALI) de cellules épithéliales bronchiques humaines par deux méthodes (Single cell-RNAseq et puces à ADN). Les résultats obtenus en Single cell-RNA seq montrent que la totalité des cellules épithéliales expriment l’ARNm CCSP. Pour pallier à ce problème, nous avons triées les cellules selon l’expression de leur protéine spécifique SCGB1A1. Les puces à ADN ont identifié trois populations exprimant la protéine CCSP (fluorescence et granulosité différentes). Ces différentes populations ont une expression d’ARNm de type ciliée ou de type classique « Club ». Ceci permet de montrer la plasticité de ces cellules et leur potentiel pour rétablir un épithélium différencié. Ensuite, nous avons montré que les voies de signalisation BMP et Notch sont déterminantes pour le devenir des cellules épithéliales des voies aériennes. Par conséquent, il est possible de maîtriser la différenciation des cellules provenant de cultures ALI NHBE et HBEC en cellules ciliées, caliciformes, Club ou basales. Un inhibiteur de Notch, le DapT, permettait de rétablir le déséquilibre caractéristique de la BPCO en favorisant le phénotype cilié, mis en évidence aussi bien en immunofluorescence qu’en single cell RNAseq. A partir des données obtenues, il est possible d’envisager de superviser le phénotype des cellules épithéliales des voies aériennes. Pour finir, nous avons étudié différents profils de sécrétions d’alarmines dans les cultures ALI de cellules épithéliales bronchiques humaines à l’état basal. TSLP et IL33 apparaissaient augmentées dans la BPCO, tandis que l’IL25 était diminuée comparé aux sujets sains et aux asthmatiques. Ces patterns d’expression étaient associés de manière hétérogène aux marqueurs cliniques de l’inflammation de type 2. Pour conclure, il n’y a donc pas une mais des cellules Club, aux morphologies, profils transcriptomiques et fonctions différentes, engagées vers des voies de différenciation distinctes. Le relatif maintien d’un profil cytokinique de type T2 d’un épithélium cultivé en ALI en relation avec les données cliniques valide ce modèle
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Quantitation of Gene Expression in Formaldehyde-Fixed and Fluorescence-Activated Sorted Cells
Article
Full-text available
Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.
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Isolation of Microarray-Quality RNA from Primary Human Cells after Intracellular Immunostaining and Fluorescence-Activated Cell Sorting
Article
Full-text available
  • Feb 2013
Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that - although the RNA isolated from these samples presented some degradation - it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis.
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Use of RNAlater in fluorescence-activated cell sorting (FACS) reduces the fluorescence from GFP but not from DsRed
Article
Full-text available
Flow cytometry utilizes signals from fluorescent markers to separate targeted cell populations for gene expression studies. However, the stress of the FACS process could change normal gene expression profiles. RNAlater could be used to stop such changes in original gene expression profiles through its ability to denature RNase and other proteins. The normal conformational structure of fluorescent proteins must be maintained in order to fluoresce. Whether or not RNAlater would affect signals from different types of intrinsic fluorescent proteins is crucial to its use in flow cytometry; this question has not been investigated in detail. To address this question, we analyzed the effect of RNAlater on fluorescence intensity of GFP, YFP, DsRed and small fluorescent molecules attached to secondary antibodies (Cy2 and Texas-Red) when used in flow cytometry. FACS results were confirmed with fluorescence microscopy. Our results showed that exposure of YFP and GFP containing cells to RNAlater reduces the intensity of their fluorescence to such an extent that separation of such labeled cells is difficult if not impossible. In contrast, signals from DsRed2, Cy2 and Texas-Red were not affected by RNAlater treatment. In addition, the background fluorescence and clumping of dissociated cells are altered by RNAlater treatment. When considering gene expression studies using cell sorting with RNAlater, DsRed is the fluorescent protein of choice while GFP/YFP have severe limitations because of their reduced fluorescence. It is necessary to examine the effects of RNAlater on signals from fluorescent markers and the physical properties (e.g., clumping) of the cells before considering its use in cell sorting.
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The RIN: an RNA integrity number for assigning integrity values to RNA measurements
Article
Full-text available
  • Feb 2006
  • BMC MOL BIOL
The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way. A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability. This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN). Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
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Fluorescence activated cell sorting (FACS) using RNAlater to minimize RNA degradation and perturbation of mRNA expression from cells involved in initial host microbe interactions
Article
Full-text available
  • Aug 2007
  • J MICROBIOL METH
Host microbe interactions frequently involve specific cellular tropism. Accurate characterization of cells involved in these initial interactions is complicated by the response to the microbe. We describe a method utilizing RNAlater for Fluorescence Activated Cell Sorting of these critical cells that minimizes the downstream perturbation in the gene expression profile.
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Isolation of full-size mRNA from cells sorted by flow cytometry
Article
  • Sep 1999
  • J BIOCHEM BIOPH METH
Gene expression is one key mechanism to regulate cell growth and differentiation. It is usually determined by Northern blotting or RT-PCR. However, studies with primary cell cultures are frequently hampered due to contaminating cells such as fibroblasts. We have developed a method to isolate intact full-size mRNA from sorted cells. In many cell types, e.g. cardiac myocytes, cell sorting without prior fixation revealed complete RNA breakdown. Based on a murine fibroblast cell line (AKR-2B), ethanol and formaldehyde at various concentrations and pre-treatment with ribonuclease inactivating DEPC were compared with each other. Fixation with 75% ice-cold DEPC–pre-treated ethanol for 5 min yielded mostly intact RNA. In contrast, antibody staining prior to sorting required 15 min fixation. Addition of RNAse-free BSA (0.5%) and 2 mM CaCl2 optimised the cell recovery ratio and thus a better RNA yield (60% compared to control) after sorting than former studies. Northern blotting and RT-PCR show the intact mRNA species β-actin. Furthermore, dependent on the cellular PCNA content, we have demonstrated the cell cycle dependent cdk2 and cyclin A expression. This fast and reliable method allows to isolate intact full-size mRNA species appropriate for Northern blotting and RT-PCR to monitor gene expression.
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Improved RNA preservation for immunolabeling and laser microdissection
Article
  • Oct 2009
  • RNA
Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.
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Erratum: Chipping away at the chip bias: RNA degradation in microarray analysis
Article
  • Jan 2004
Measurement of gene expression is based on the assumption that an analyzed RNA sample closely represents the amount of transcripts in vivo. Transcripts show stability differences of up to two orders of magnitude in vivo1, raising the possibility that partial degradation during cell lysis could cause a variable extent of bias in quantification of different transcripts. One of the most effective tools for characterizing RNA integrity is capillary electrophoresis, in which RNA degradation is indicated by an altered 28S/18S rRNA signal ratio2. In the software of the commonly used system (Bioanalyzer 2100, Agilent), quantification of 18S and 28S rRNA is compromised by the fact that this calculation is based on area measurements that are heavily dependent on definitions of start and end points of peaks (Fig. 1a). Even accurate determination of this ratio is not sufficient to detect degradation efficiently (Fig. 1b). We developed a mathematical model that results in an objective number for quantitative characterization of RNA degradation. Aside from three prominent peaks (small RNAs, 18S and 28S rRNA), a chromatogram of the size distribution of cellular RNAs shows a broad range of molecular weights with much weaker signals. With increasing degradation, heights of 18S and 28S peaks gradually decrease and additional 'degradation peak signals' appear in a molecular weight range between small RNAs and the 18S peak (Fig. 1b). The ratio of the average degradation peak signal to the 18S peak signal multiplied by 100 will hereafter be referred to as the degradation factor. This analysis has been tested on 19 tissues of seven organisms, and it is a reproducible parameter for degradation of mammalian RNA (Supplementary Table 1 online). As an example, 12 repeated measurements of the same sample yielded an average degradation factor of 27.14 with a standard deviation of 1.06. Degradometer software for calculation of the degradation factor can be downloaded from http://www.dnaarrays.org.
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RNA integrity and the effect on the real-time qRT-PCR performance
Article
  • Apr 2006
  • MOL ASPECTS MED
The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. Using intact RNA is a key element for the successful application of modern molecular biological methods, like qRT-PCR or micro-array analysis. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment. Profiles generated yield information on RNA concentration, allow a visual inspection of RNA integrity, and generate approximated ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for the qRT-PCR was analyzed by determining the RNA quality of different bovine tissues and cell culture. Independent analysis systems are described and compared (OD measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Further the comparison and correlation between the total RNA integrity on PCR performance as well as on PCR efficiency is described. On the basis of the derived results we can argue that qRT-PCR performance is affected by the RNA integrity and PCR efficiency in general is not affected by the RNA integrity. We can recommend a RIN higher than five as good total RNA quality and higher than eight as perfect total RNA for downstream application.
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Kornacker Chipping away at the chip bias: RNA degradation in microarray analysis
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