Successful measurement of gene expression by quantitative ... · cDNA synthesis random primer vs....
Transcript of Successful measurement of gene expression by quantitative ... · cDNA synthesis random primer vs....
27.03.07 F:\...\070327_Talk qPCR Meeting Munich (qPCR + FFPE).ppt 1
Successful measurement of gene expression by
quantitative PCR and DNA chip analysis
with RNA derived of FFPE material
Rolf Jaggi
Department of Clinial Research Research - Univ. of Bern
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Contents of talk
• Material kryo-preserved 4x 25 µm thick sectionsFFPE material 5-10x 10 µm thick sections
• Methods RNA Isolation "own" protocolhomogenize in lysis bufferdigest proteinase KDe-modify chemical modifications induced by formalin
cDNA synthesis random primer vs. gene-specific primers
QPCR short ampliconsMGB assays or LNA assay
DNA chip experiments "own" primers for cDNA synthesisArrays CombiMatrix 12k
Agilent 44k
• Results QPCR single genesprofiles (expression of multiple genes)
DNA chips kryo vs. FFPEsingle genesprofiles
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Procedure/major steps for QPCR-based analyses
• Identification of marker genes from microarray studies, literature etc.
• Reduce number of test genes to minimum
• Select appropriate control genes
• Isolate RNA from FFPE sections
• Establish robust real-time assays which give reproducible results even with fragmented RNA
• Evaluate results on the basis of single genes
• Develop model (= scoring system) involving groups of genes, we used
Estrogen scoreHER2 score Prognostic scoreProliferation score
Cronin et al. Am J Pathol 164 (2), 35-42, 2002. Measurement of Gene Expression in Archival Paraffin-Embedded Tissues.
⇒ 16 test genes & 5 control genes
⇒ Recurrence Score (RS)→ prognostic for patients with primary
breast cancer (stage I or II, N0, ER+)commercial test is available (~3'000$ per test)
e.g. Oncotype DX
⇒ RS is also predictive for response to chemotherapy
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Experimental design
14 breast cancers
kryo-preserved sample
isolate total RNA = A (standard procedure)
FFPE block
isolate RNA⇒ B, Qiagen FFPE protocol⇒ C, ncLysis buffer (Applied Biosystems)⇒ D, "own" procedure (silica-based spin columns)
"matched samples"
De-modify
make gene-specific cDNATaqMan PCR (MGB assays or LNA probes)Evaluation:
Expression analysis of individual genesNormalizationDevelop models (= scoring system) of multiple genes
Estrogen-response scoreProliferation scorePrognostic score
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Experimental design
14 breast cancers
make gene-specific cDNATaqMan PCR (MGB assays or LNA probes)Evaluation:
Expression analysis of individual genesNormalizationDevelop models (= scoring system) of multiple genes
Estrogen-response scoreProliferation scorePrognostic score
kryo-preserved sample
isolate total RNA = A (standard procedure)
FFPE block
isolate RNA⇒ B, Qiagen FFPE protocol⇒ C, ncLysis buffer (Applied Biosystems)⇒ D, "own" procedure (silica-based spin columns)
"matched samples"
De-modify
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QPCR
single gene data
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RNA quality is critical: comparison of different protocols
0.2
0.5
1.0
2.0
4.0
6.0
kryo
A =
Qia
gen
B =
ncL
ysis
D =
ow
n
M (l
adde
r)
kbGAPDH
(gene-specific, single-primer)
10
15
20
25
30
35
40
2 3 6 8 10 13 14 15 16 17 18 19 20 22
cDNA
Ct
ABCD
A = kryo B = QiagenC = ncLysisD = own
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Comparison of own protocol with different commercial kits Stratagene, RecoverAll (Ambion)
GAPDH
23
25
27
29
4 1 0.25
ng RNA input
Ct
De-mod. (3)De-mod. (1)De-mod. (2)RecoverAll Stratagene FFPE Kit
IGBP5
22
24
26
28
30
4 1 0.25
ng RNA input
Ct
De-mod. (1)De-mod. (2)De-mod. (3)RecoverAll Stratagene FFPE Kit
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Comparison of results with different de-modification protocols (1)
24
28
32
36
40
no (1) (2) (3)
RNAlater De-Modification
Ct
IGBP5 longIGBP5 mediumIGBP5 short
no (1) (2) (3)
FFPE – De-modification
20
24
28
32
36
40
44
no (1) (2) (3)
RNAlater De-ModificationC
t
GUSRPLPOTFRCRPLP0
no (1) (2) (3)
FFPE – De-modification
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Comparison of estrogen receptor expression and results based on immunohistochemistry
Tumor #
ER-status (IHC)0
5
10
15
20
25
30
15 18 20 10 6 22 2 8 16 13 14 17 3 19
neg neg neg + + + + + + + + + + +
arbi
trar
y un
its (l
og2)
kryo (A_gs)
FFPE (D_gs)
ESR1 expression
AB: MGB assays
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QPCR
Multiple genes ⇒ Scores
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ERBB2 IHC = 3+
ERBB2 IHC = 3+
IHC = -ve IHC = -ve
Test Rank corr. p-valueA_gs vs D_gs 0.95 < 0.001
FFPE-derived materialkryo-preserved tumor material
Proliferation score: results from 14 tumors
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ER score ER score ER score (based on mean) (based on median) (based on geometric mean)
Normalization based on reference genes
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Array data
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QPCR DNA chips
– Limited number of features/experimentHigh sensitivity of methodWorks with degraded RNA ( used with optimized reverse transcription and qPCR assays)Large number of samples potentially available from clinical trials
– No standards for normalization
Large number of features/arrayMethod depends on good quality RNA (e.g. kryo)Established procedures fornormalization
– Samples are oftenheterogeneous
– Limited number of samples per study
– Various treatment regimens– Outcomes sometimes unknown
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Application of FFPE-derived RNA to DNA chips
fragmented RNA
FFPE material(ZR-75-1 cells)
DNA chipCombiMatrix
DNA chipCombiMatrix
cRNA modified prot.
cRNA standard prot.
intact RNA
Kryo material (ZR-75-1 cells)
DNA chipCombiMatrix
DNA chipCombiMatrix
cRNA modified prot.
cRNA standard prot.
compare kryo vs. FFPE (standard)
modified
technicalreplicates
technicalreplicates
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53785_#4b vs 4b
4
6
8
10
12
14
16
4 6 8 10 12 14 16
53785_#4a vs. 4a
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6
8
10
12
14
16
4 6 8 10 12 14 16
CombiMatrix 12k chips: Technical replicates
standard protocol
modified protocol
Correlation: ~0.4 – 0.58
Correlation: ~0.98
technicalreplicates
technicalreplicates
compare kryo vs. FFPE
standard prot.
modified prot.
compare kryo vs. FFPE
kryo
FFPE
kryo
FFPE
Correlation: 0.82 – 0.87
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Tumor #2kryo vs. FFPE, standard protocol
Tumor #2 kryo vs. FFPE, modified protocol
Agilent chips: kryo vs. FFPE tumor RNA (Scatterplot)
Tumor #2kryo, standard protocol
Tum
or #
2FF
PE
, sta
ndar
d pr
otoc
ol
Tumor #2kryo, modified protocol
Tum
or #
2FF
PE
, mod
ified
pro
toco
l
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ER Expression in 6 tumors
ER Status + + + - + + + + + - + +
FFPE kryo/RNAlaterDNA chip
QPCR results
0
5
10
15
20
Tu#1 Tu#2 Tu#3 Tu#4 Tu#5 Tu#6 Tu#1 Tu#2 Tu#3 Tu#4 Tu#5 Tu#6
FFPE kryo
Arb
itra
ry u
nit
s (
log
2)
ESR1GAPDHRPL7ARPLPOUBB
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0.9120.9140.9240.9440.8930.4510.9470.4680.9410.779Spearman(ranks) (116)
0.930.9180.9360.920.8890.4590.8970.5810.9620.923Pearson (116)
Tu #6modif.
Tu #5modif.
Tu #4modified
Tu #3modified
Tu #2modified
Tu #2standard
Tu #1modified
Tu #1standard
ZR-75-1modified
ZR-75-1standard
Summarization of results: correlations
Agilent 44k chips 60-mer oligosall genes 41'00021 Paik genes represented by 116 spots on the chip
intact vs. fragmented RNA fom cells
kryo vs. FFPE tumor RNAfrom breast cancer
0.8350.8230.8370.7930.8140.5630.8170.5850.9050.935Spearman(ranks) (all genes)
0.8620.8570.8620.8290.8380.6220.8450.6610.9080.932Pearson (all genes)
Tu #6modif.
Tu #5modif.
Tu #4modified
Tu #3modified
Tu #2modified
Tu #2standard
Tu #1modified
Tu #1standard
ZR-75-1modified
ZR-75-1standard
41'000 genes
21 Paik genes
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• Material- kryo material 4x 25µm thick sections QPCR/DNA chip
feasible
- FFPE Material 5-10x 10µm sections QPCRtechnically demandingDNA chip
feasible specialized protocols
• Technology- RNA Isolation Homogenization
Proteinase K digestionDe-modification
- cDNA synthesis Gene-specific primers during RT
- QPCR short ampliconsMGB assays or LNA assays
Summary
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• Janine AntonovSybille MattheyAndrea OberliAnna Baltzer
• Hans Jörg Altermatt, MD Pathology Länggasse, CH-3012 Bern
• Achim Fleischmann, MD
• Vlad Popovic, PhDMauro Delorenzi, PhD
• Support: Bernese Cancer League (BKL)Swiss Cancer League (SKL)International Breast Cancer Study Group (IBCSG)Applied Biosystems, Rotkreuz, Switzerland
Collaborators