a novel and universal method for microrna rt-qpcr data ... novel and universal method for microrna...
TRANSCRIPT
![Page 1: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/1.jpg)
A novel and universal method for microRNA RT-qPCRdata normalization
Jo Vandesompele
professor, Ghent University
co-founder and CEO, Biogazelle
4th International qPCR Symposium
Weihenstephan, March 10, 2009
![Page 2: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/2.jpg)
outline
megaplex stem-loop RT-PCR & PreAmp preamplification
normalization of microRNA gene expression levels
![Page 3: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/3.jpg)
microRNA quantification platform
hybridisation based (microarray or beads in solution)
Exiqon probeset | miRCURY arrays | flexmiR beads
Ambion mirVana probeset
Invitrogen NCode probeset
Agilent Human miRNA Microarray
Asuragen DiscovArray (service)
PCR based
Applied Biosystems stem-loop RT-PCR
Exiqon miRCURY LNA microRNA PCR System
Invitrogen NCodemiRNA RT-PCR
Qiagen miScript primer set
miQPCR and other home brew protocols
sequencing based (RNAseq)
![Page 4: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/4.jpg)
microRNA expression profiling
stem-loop megaplex reverse transcription using 20 ng total RNA
limited-cycle pre-amplification
qPCR profiling 450 miRNAs and controls
higher sensitivity
minimal amplification bias (Mestdagh et al., Nucleic Acids Research, 2008)
profiled > 1000 samples
![Page 5: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/5.jpg)
platform evaluation
RT-qPCR
qPCR plate setup: gene maximization (Hellemans et al., Genome
Biology, 2007)
o a different miRNA in each well of a 384 well plate (no replicates)
o 1 sample per 384 well plate
sample input
o 20 ng total RNA (PreAmp)
o 1.6 µg total RNA
liquid handling Tecan Evo
qPCR reactions on 7900HT
quality control
o mean Cq for each 384 well plate
o number of not expressed miRNAs
![Page 6: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/6.jpg)
0
1
2
3
4
5
6
7
8
10 15 20 25 30 35
0
0.5
1
1.5
2
2.5
10 15 20 25 30
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0
1
2
3
4
5
6
7
8
10 15 20 25 30 35
Average CqNP (NBL-S, IMR-32) Average CqNP (NBL-S, IMR-32)∆∆
Cq
(|∆
Cq
NP
-∆
Cq
P|)
NB
L-S
, IM
R-3
2A
ve
rag
e ∆
∆C
q
∆∆
Cq
(|∆
Cq
NP
-∆
Cq
P|)
NB
L-S
, IM
R-3
2A
ve
rag
e ∆
∆C
q
minimal pre-amplification bias
![Page 7: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/7.jpg)
0
5
10
15
20
25
30
0 2 4 6 8 10 12 14
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
0
5
10
15
20
25
30
0 2 4 6 8
0
5
10
15
20
25
30
35
0 2 4 6 8
Cq
-va
lue
Cq
-va
lue
Cq
-va
lue
Cq
-va
lue
total cell number total RNA input (pg)
miR-18a
R2 = 0.975
miR-20b
R2 = 0.993
miR-92
R2 = 0.998
miR-19a
R2 = 0.996
A B
1 2 4 8 16 32 64 128
1 2 4 8 16 32 64 128
0 2 4 6 8 10 12 14
0 2 4 6 8 10 12 14
30
25
20
15
10
5
0
30
25
20
15
10
5
0
35
30
25
20
15
10
5
0
35
30
25
20
15
10
5
0
single cell profiling
total cell number total RNA input (pg)
![Page 8: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/8.jpg)
Mestdagh et al., Nucleic Acids Research, 2008
![Page 9: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/9.jpg)
normalization
removal of experimentally induced noise
input quantity: RNA quantity, cDNA synthesis efficiency, …
input quality: RNA integrity, RNA purity, …
gold standard is the use of multiple stably expressed reference genes
which genes?
how many?
how to do the calculations?
![Page 10: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/10.jpg)
geNorm normalisation
framework for qPCR gene expression normalisation using the reference gene concept:
quantified errors related to the use of a single reference gene
(> 3 fold in 25% of the cases; > 6 fold in 10% of the cases)
developed a robust algorithm for assessment of expression stability of candidate reference genes
proposed the geometric mean of at least 3 reference genes for accurate and reliable normalisation
Vandesompele et al., Genome Biology, 2002
> 1250 citations in PubMed
> 8000 software downloads
![Page 11: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/11.jpg)
http://medgen.ugent.be/genorm
geNorm software
automated analysis
ranking of candidate reference genes according to their stability
determination of how many genes are required for reliable normalization
![Page 12: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/12.jpg)
robust – insensitive to outliers
maximal reduction of experimental variation
accurate assessment of small expression differences
statistically more significant results
geNorm validation
![Page 13: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/13.jpg)
microRNA normalization
small-RNA controls
classic normalization strategy
small nuclear RNAs, small nucleolar RNAs
18 available from Applied Biosystems
mean normalization
method applied for microarray data
universal: applicable for every miRNA dataset
many datapoints needed (megaplex vs. multiplex)
miRNAs/controls that resemble the mean
minimal standard deviation when comparing miRNA expression with mean ( geNorm V value, st dev of log transformed ratios)
compatible with multiplex asays
need to determine mean
![Page 14: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/14.jpg)
small RNA controls
How ‘stable’ is the mean compared to controls?
geNorm analysis using controls and mean as input variables
exclusion of potentially co-regulated controls
HY3 7q36
RNU19 5q31.2
RNU24 9q34
RNU38B 1p34.1-p32
RNU43 22q13
RNU44 1q25.1
RNU48 6p21.32
RNU49 17p11.2
RNU58A 18q21
RNU58B 18q21
RNU66 1p22.1
RNU6B 10p13
U18 15q22
U47 1q25.1
U54 8q12
U75 1q25.1
Z30 17q12
RPL21 13q12.2
![Page 15: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/15.jpg)
miRNA expression datasets
neuroblastoma tumour samples
T-ALL samples
EVI1 deregulated leukemias
retinoblastoma tumour samples
normal tissues
normal bone marrow
![Page 16: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/16.jpg)
0
0,2
0,4
0,6
0,8
1
1,2
1,4
exp
ressio
n s
tab
ility
neuroblastoma
![Page 17: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/17.jpg)
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
exp
ressio
n s
tab
ility
T-ALL
![Page 18: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/18.jpg)
0
0,5
1
1,5
2
2,5
exp
ressio
n s
tab
ility
normal tissues
![Page 19: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/19.jpg)
0
20
40
60
80
100
120
0 50 100 150 200 250 300
not normalised
stable controls
mean
miRNAs
neuroblastoma
![Page 20: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/20.jpg)
0
10
20
30
40
50
60
70
80
not normalised stable controls mean miRNAs
neuroblastoma
![Page 21: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/21.jpg)
0
20
40
60
80
100
120
0 50 100 150 200 250 300
not normalised
stable controls
mean
miRNAs
T-ALL
![Page 22: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/22.jpg)
0
10
20
30
40
50
60
70
80
90
100
not normalised stable controls mean miRNAs
T-ALL
![Page 23: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/23.jpg)
0
20
40
60
80
100
120
0 50 100 150 200 250 300
not normalised
stable controls
mean
miRNAs
normal tissues
![Page 24: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/24.jpg)
0
10
20
30
40
50
60
70
80
not normalised stable controls mean miRNAs
normal tissues
![Page 25: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/25.jpg)
biological validation
MYCN binds to the mir-17-92 promoter
CpG island
mir-17-92 cluster
+5 kb-5 kb
CA
TG
TG
CA
TG
TG
CA
TG
TG
CA
CG
TG
CA
CG
TG
CA
TG
TG
CA
TG
TG
A B C
0123456789
101112
A B C
Fold
en
rich
men
t
Amplicon
IMR5
WAC2
![Page 26: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/26.jpg)
biological validation
choice of normalization strategy influences differential miRNA expression
Mir-17-92 expression in neuroblastoma tumours
0
0,5
1
1,5
2
2,5
3
3,5
stable controls
mean
miRNAs
![Page 27: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/27.jpg)
biological validation
choice of normalization strategy influences differential miRNA expression
Mir-17-92 expression in neuroblastoma tumours
0
0,5
1
1,5
2
2,5
3
3,5
stable controls
mean
miRNAs
![Page 28: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/28.jpg)
biological validation
choice of normalization strategy influences differential miRNA expression
Mir-17-92 expression in neuroblastoma tumours
0
0,5
1
1,5
2
2,5
3
3,5
stable controls
mean
miRNAs
![Page 29: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/29.jpg)
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
fold
ch
an
ge
(M
YC
N a
mp
lifie
d v
s. M
YC
N s
ing
le c
op
y)
controls
mean
average FCcontrols = -0.404average Fcmean = 0.050average FCmiRNAs = 0.124
balanced differential expression
![Page 30: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/30.jpg)
conclusions
a highly sensitive miRNA expression profiling platform
novel and powerful miRNA normalization strategy
maximal reduction of technical noise
improved identification of differentially expressed genes
balancing of differential expression
universally applicable
o mean
o multiple stable endogenous controls
![Page 31: A novel and universal method for microRNA RT-qPCR data ... novel and universal method for microRNA RT-qPCR data normalization Jo Vandesompele professor, Ghent University ... RT-qPCR](https://reader031.vdocuments.us/reader031/viewer/2022011820/5ea5492898234449ab229a58/html5/thumbnails/31.jpg)
acknowledgments
miRNA, T-UCR
Pieter Mestdagh
Frank Speleman
Applied Biosystems
qbasePLUS
Jan Hellemans
Stefaan Derveaux
RNA QC, RNA amplification
Nurten Yigit
Justin Nuytens
SHEP-tet-17-92
Johannes Schulte (Essen)
MYCN-ChIP
Frank Westerman (Heidelberg)