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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
"which was partially alleviated by lowering to a half the amount of input rna"
symptom
hodgkin lymphoma, classical
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