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Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas.
[hodgkin lymphoma, classical]
Formalin-fixed
paraffin-embedded
(
FFPE
)
tissues
are
invaluable
sources
of
biological
material
for
research
and
diagnostic
purposes
.
In
this
study
,
we
aimed
to
identify
biological
and
technical
variability
in
RT-qPCR
TaqMan
®
assays
performed
with
FFPE-RNA
from
lymph
nodes
of
classical
Hodgkin
lymphoma
samples
.
An
ANOVA-nested
6
-
level
design
was
employed
to
evaluate
BCL
2
,
CASP
3
,
IRF
4
,
LYZ
and
STAT
1
gene
expression
.
The
most
variable
genes
were
CASP
3
(
low
expression
)
and
LYZ
(
high
expression
)
.
Total
variability
decreased
after
normalization
for
all
genes
,
except
by
LYZ
.
Genes
with
moderate
and
low
expression
were
identified
and
suffered
more
the
effects
of
the
technical
manipulation
than
high
-expression
genes
.
Pre-amplification
was
shown
to
introduce
significant
technical
variability
,
which
was
partially
alleviated
by
lowering
to
a
half
the
amount
of
input
RNA
.
Ct
and
Cy
0
quantification
methods
,
based
on
cycle-threshold
and
the
kinetic
of
amplification
curves
,
respectively
,
were
compared
.
Cy
0
method
resulted
in
higher
quantification
values
,
leading
to
the
decrease
of
total
variability
in
CASP
3
and
LYZ
genes
.
The
mean
individual
noise
was
0
.
45
(
0
.
31
to
0
.
61
SD
)
,
indicating
a
variation
of
gene
expression
over
~
1
.
5
folds
from
one
case
to
another
.
We
showed
that
total
variability
in
RT-qPCR
from
FFPE-RNA
is
not
higher
than
that
reported
for
fresh
complex
tissues
,
and
identified
gene
-
,
and
expression
level-sources
of
biological
and
technical
variability
,
which
can
allow
better
strategies
for
designing
RT-qPCR
assays
from
highly
degraded
and
inhibited
samples
.
Diseases
Validation
Diseases presenting
"high expression"
symptom
carcinoma of the gallbladder
cholangiocarcinoma
esophageal adenocarcinoma
esophageal carcinoma
esophageal squamous cell carcinoma
gm1 gangliosidosis
hodgkin lymphoma, classical
malignant atrophic papulosis
phenylketonuria
primary effusion lymphoma
severe combined immunodeficiency
waldenström macroglobulinemia
wiskott-aldrich syndrome
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