Transcription

Conditions de fiabilité
des mesures d’expression génique par RT-qPCR
en cancérologie clinique
J.D. Combes1,2, G. Grelier3, M. Laversanne2, N. Voirin1,
S. Chabaud1, R. Ecochard2, C. Moyret-Lalle3 & C. Lasset1
1
Département de santé publique, Centre Léon Bérard, Lyon
2 Service de biostatistiques des Hospices Civils de Lyon, CNRS UMR 5580
3 INSERM U590, Centre Léon Bérard, Lyon
PCR, qPCR, RT-qPCR...

Polymerase Chain Reaction

Real-Time quantitative PCR

Reverse-Transcription Real-Time quantitative PCR
Transcription
reverse (ADNc)
qPCR
?
2/10
RT-qPCR en cancérologie
RT-qPCR
Extraction
Transcription
ARN
ADNc
qPCR
Cancer
T47D
Culture
cellulaire
Expression
APC11
APC11+??
Non
MB45
Cancer
9
Culture
cellulaire
RT-qPCR
Extraction
Transcription
ARN
ADNc
qPCR
Expression
APC11
APC11-??
3/10
Plan expérimental
T47D
Culture
cellulaire
Extraction Transcription
ARN
ADNc
qPCR
MB45
9
4/10
Plan expérimental
BT-20
MCF-7
MDA-MB459
BT-474
MDA-MB157
MDA-MB549
Cal51
MDA-MB231
SK-Br3
Cama1
MDA-MB361
T47D
HCC-1937
MDA-MB436
UACC-612
HS-57BT
MDA-MB453
ZR-75.1
5/10
Estimation de l’erreur de mesure
Lignées cellulaires (18)
18
3
2
1
Source de variabilité
Culture
cellulaire
Extraction
ARN
Transcription
ADNc
Inter lignée
ET
?
Culture
?
Extraction ARN
?
RT
?
qPCR
?
qPCR
6/10
Lignées cellulaires (18)
18
3
2
1
Culture
cellulaire
Extraction
ARN
Transcription
ADNc
Source de variabilité
ET
Inter lignée
1.7
Culture
1.3
Extraction ARN
1.2
RT
2.0
qPCR
0.26
qPCR
7/10
?
?
?
?
8/10
Conclusion
Fiabilité des mesures d’expression génique?
Murphy J, Bustin SA.
Reliability
reverse-transcription
PCR
in clinical
diagnostics:
clinical
diagnostics:
Tichopadof
A,real-time
Kitchen R,
Riedmaier I, Becker C,
Ståhlberg
A, Kubista
M.
PfafflorMW.
gold
standard
substandard?
Design and
optimization
of
reverse-transcription
quantitative PCR experiments.
PCR
Étapes pré-The
ongoing
evolutionReview.
of qPCR.
Expert Rev
Mol
Diagn.
2009
Mar;9(2):187-97.
PubMed
PMID: 19298142.
Clin
Chem.
2009
Oct;55(10):1816-23.
PubMed
PMID: 19643838.
2009
Oct;55(10):1816-23.
Methods. 2010 Apr;50(4):215-6. PubMed PMID: 20215019.
recommend
the use of issample
replicates
““We
However,
this technology
still troubled
by
preferentially
to any other
replicates
significant
technical
deficiencies.
Publication
of
Quantitative
Real
PCR) and RT
pre-PCR steps like sampling, nucleic acid-Time
stabilisation
when working
with
solid tissue, cell
and single
Hence its
often-improper
use culture,
as a clinical
tool cells,
are still highly variable and
and wehas
recommend
the use
of RT replicates
public health
implications,
introduce lots of error in the quantification procedure.”
when working
with blood.”
most recently demonstrated
through
its association with the
measles, mumps and rubella vaccine/autism controversy.”
recommend
the
use of issample
replicates
““We
However,
this technology
still
troubled
by
MIQE
Guidelines
(Minimum
Information

“Most advances were implemented in PCR method for
itself but
9/10
Remerciements
INSERM U590 - CLB
Dépt de Santé Publique - CLB
Biostatistiques HCL
Gaël Grelier
Caroline Moyret-Lalle
Alain Puisieux
Sylvie Chabaud
Christine Lasset
David Perol
Nicolas Voirin
René Ecochard
Jean Iwaz
Matthieu Laversanne
Pascal Roy
Fabien Subtil
Références
1: Combes JD, Grelier G, Laversanne M, Voirin N, Chabaud S, Ecochard R, Lasset C, Moyret-Lalle C. Contribution of
cell culture, RNA extraction, and reverse transcription to the measurement error in quantitative reverse transcription
polymerase chain reaction-based gene expression quantification. Anal Biochem. 2009 Oct 1;393(1):29-35. Epub 2009 Jun
13. PubMed PMID: 19527676.
2: Murphy J, Bustin SA. Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or
substandard? Expert Rev Mol Diagn. 2009 Mar;9(2):187-97. Review. PubMed PMID: 19298142.
3: Pfaffl MW. The ongoing evolution of qPCR. Methods. 2010 Apr;50(4):215-6.PubMed PMID: 20215019.
4: Tichopad A, Kitchen R, Riedmaier I, Becker C, Ståhlberg A, Kubista M. Design and optimization of reversetranscription quantitative PCR experiments. Clin Chem. 2009 Oct;55(10):1816-23. Epub 2009 Jul 30. PubMed PMID:
19643838.
10/10
8
+
BT
_4
74
+
T4
7D
_1
93
7
5
BT
_2
0
C
+
C
am
a_
1
+
C
75
.1
+
ZR
M
B1
57
F_
7
+
M
D
A_
+
M
C
4
H
81
2
M
B2
31
C
+
M
D
A_
AC
+
U
+
al
_5
1
3
C
M
B3
61
Br
3
+
M
D
A_
+
SK
+
M
B4
53
M
B4
59
+
M
D
A_
2
M
D
A_
S_
57
8T
+
H
M
B4
36
M
B5
49
1
M
D
A_
M
D
A_
Gene expression value
Données
16
16
15
15
14
14
13
13
12
12
11
11
10
10
9
9
+
7
6
+
+
8
7
6
5
4
3
2
1
Erreur
de
mesure
due
Erreur
de
mesure
due
Variabilité
ààla
Reverse
Transcription
l’extraction
de
l’ARN
la culture cellulaire
interlignées
cellulaires