Download Personalized Genetic Technology and Novel Multiplex Quantitative

Personalized Genetic Technology and
Novel Multiplex Quantitative PCR
Hong Kong University
Tiffany Jiang Ph.D.
Asia Pacific Marketing Manager
Beckman Coulter
At your fingertip!!
Multiplex Gene Expression
Sequencing
STR
SNP
AFLP
T-RFLP
Methylation
Other Fragment Analysis
Gene Expression
Cell has ~30,000 genes
On average ~ 10,000 expressed in a cell
10 to 500 associated with a particular disease
10 to 50 associated with a pathway or response
10,000
1000
100
Signatures
10
# of Genes
Disc
overy
100
1000
10,000 100,000
# of Samples
Disease Signature
DrugResponse
Response
Drug
Cancer Prognosis
• Cytotoxicity and DNA damage
• • Anticancer
drug
HepG2 cell
lineresponse
study
• • Human
ID’d a tumor/xenograft
20 gene set out model
of 250
• • 1570
genes 100
differentially
respond
Classified
compounds
correctly
cytotoxic
anti-inflammatory
and
• 32asgene
signature
tracked potency
•
•
•
•
•
damaging
agents
ofDNA
response
to 7 chemotherapy
agents
Burczynski et al.; Tox. Sci. 58,
H. Zembutsu et.al. Cancer Res 62,
399-415 (2000)
518-27 (2002)
Breast cancer
Human clinical breast cancer study
ID’d a 70 gene set out of 25,000
Prognostic signature for metastases
Outperformed other clinical parameters in
predicting disease outcome
van’t Veer et al., Nature 415,
530-536 (2002)
Assessing Glioblastoma Prognosis Using A
Multiplex RT-PCR Assay on Formalin Fixed Paraffin
Embedded (FFPE) Tissue
Tumor grade and survival of gliomas
•The influence
of tumor grade
on survival is a
well-recognized
prognostic
feature of
gliomas.
38 genes appear robustly associated
with survival
Actual survival data
Scrambled survival data
38 genes: survival in all 4 data sets
(MD, UCSF, UCLA, MGH)
FFPE
•• Test
Test needs
needs to
to be
be amenable
amenable to
to routinely
routinely
processed,
processed, clinically
clinically available
available tissue:
tissue:
–– Formalin
Formalin fixed,
fixed, paraffin
paraffin embedded
embedded
specimens
specimens
1000
Discovery
Arrays
100
10
# of Genes
10,000
Accelerate: Multiplex PCR
Signatures
Multiplex PCR
(eXpress Profiling)
RT PCR
100
1000
10,000 100,000
# of Samples
• eXpress Profiling (XP PCR)
• Multiplexed low-cost process
(2-40 genes)
• High sensitivity (104 molecules)
• 3+ log dynamic range
• Low sample requirement (5 ng)
• Work with small tissue samples
• Work with varied quality RNA
(fixed samples)
• Endpoint PCR with capillary
readout
Key Components of GeXP
• Chemistry (XP-PCR)
– Patented priming strategy
• PCR- based
• Maintains relative gene ratios
• Minimizes PCR artifacts
•
•
•
•
Multiplexed low-cost process (2-40 genes)
Solution-phase RT-PCR
High sensitivity
Low sample requirements
– As little as 5 ng total RNA/reaction
– Work with small clinical samples
Process Overview
RT
Gene-specific
Multiplex
XP-PCR
Total RNA
96-well format
Sample dilution
Gene Expression
Analysis
XP PCR Priming Process
• 1: Reverse Transcription mRNA to cDNA
– Reverse Chimeric Primers priming specific transcripts
in Total mRNA preparation.
Universal Reverse Tag sequence
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Gene Specific Primers
XP PCR Priming Process
• 2a Multiplex PCR
– Step 1 of the reaction:
• Universal/Gene specific primers
• Cycle 1 to 3- gene specific primers work
• Set gene ratios
Forward Chimeric Primer with
Universal Forward Primer Sequence
Gene 1 cDNA
Gene 2 cDNA
Gene 3 cDNA
Universal Reverse Primer sequence added in RT step
XP PCR Priming Process
• 2b Multiplex PCR
– Cycles 3 through 35
– Universal primer in excess concentration take over- and amplify
multiple target fragments simultaneously and uniformly.
– Treats all templates as same chemical entity
[
]
Universal reagents in reaction are in
excess, driving amplification.
Dye-Labeled Forward Universal Primer
Unlabeled Universal Primer
After Universal Amplification
HuMulti2
SKL.A10_041223080B
80000
• Capillary Electrophoresis
Separation of Fragments
– by size
– precise signal
quantification.
• No clean-up!
60000
157.70
50000
325.74
40000
172.03
30000
208.66
303.47
178.37
20000
235.75
249.93
10000
272.83
152.03
221.34
194.02
Dye Signal
Unincorporated primers and
fragments diluted, separated, data
filtered, and ready for analysis.
214.60
70000
163.69
186.76
242.76
202.13
286.14 294.16
280.78
255.07
265.64
227.33
314.63
340.69
352.33
0
150
175
200
225
250
Size (nt)
275
300
325
350
{
{
{
GenomeLab™ GeXP Genetic Analysis System
• Size range 150-350 nt
40000
beta actin
277.96
35000
cyclophilin A
307.03
30000
341.64
• amplification products are well
balanced within a linear detection
range of the CE system for precise
signal quantification
25000
• Reference genes are included to
allow peak signal normalization for
relative quantitative analysis
314.28
20000
172.82
200.44
264.43
164.85
285.90
333.68
15000
210.05
10000
150.90
GAPDH
245.69
181.13
224.25
272.05
250.07
• Gene-specific peaks -identified,
quantitated and normalized
298.97
290.92
324.73
228.51
5000
158.46
238.99
195.68
171.65
257.25
188.55
276.87
305.89
349.81
0
150
175
200
225
250
Size (nt)
275
300
325
350
• Gene expression profiles - created
and visualized using proprietary
software.
FFPE Assay Design
• short amplicons: <75 bp
• variable length in chimeric primers
• performed and optimized by Althea, Inc.
• two multiplexes - total 24 genes
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Chimeric
Chimeric primers
primers
HuMulti2
SKL.A10_041223080B
80000
Kan(r)
Kan(r)
60000
214.60
Separation between
between peak
peak {{
Separation
Minimum Size
Size {{
Minimum
70000
157.70
50000
40000
172.03
208.66
178.37
20000
Maximum Size
Size {{
Maximum
30000
325.74
303.47
235.75
249.93
10000
272.83
286.14
152.03
294.16
221.34
280.78
194.02
255.07
242.76
163.69
Dye Signal
265.64
227.33
202.13
186.76
352.33
314.63
340.69
0
150
175
200
225
250
Size (nt)
275
300
325
350
Developing ’38 genes’ into a useful test
• develop score: summarize expression
profile into a single number (like PSA
score)
• simplify: reduce genes from 38
• adapt: for clinical use needs to
work with clinical samples
• accelerate: to achieve sufficient
throughput for clinical use
• validate: test in independent
sample set
• clinical samples being treated with
current standard of care
Case VII: Single Cell Analysis
Automation
Flow
GeXP
AmpliGrid
̶
Single cell gene expression profiling
Detecting multiple genes from a single cell
without pre-amplification
HuRef Multiplex on
Breast cancer cell
No pre-amplification, no bias in the final results
Fluidigm system pre-amplify targets with 35 cycles
Induced
Pluripotent
Stem cell
Skin cells
Isolation
Patients
Genetic Diseases
iPS
Reprogramming
Expansion
Personalized Stem
Cell Therapies
Differentiation
Autologous
Transplantation
hC
C
N
hG G1
AP
D
H
hP
H
B
hT
BP
hc
M
yc
hK
lf4
hO
ct
hS 4
O
X
hN 2
an
og
hL
hD in2
N 8
M
T
hD 3B
PP
hD A2
PP
A
hG 4
D
F
hG 3
U
S
hM B
YB
L
hP 2
H
C
hS 1
A
hS LL4
TE
L
hT LA
ER
Tv
hZ 1
M FP4
Xs
2
M hOc
Xs
t4
-h
So
M
X s x2
h
M
Xs Klf4
M -hc
Xs M
-h y c
M Nan
Xs
o
-h g
LI
N
28
Normalized Values
Automation
12
10
CA-1 hESC
BJ-pMXs
4YA hiPSC
GeXP
8
6
4
2
0
Flow
Vehicle Control
3
Rosiglitazone 100 uM
Troglitazone 100 uM
2
*
1
Genes
B Ppi
et
a
aac
tin
4
H
o1
C
yp
1A
1
C
yp
1A
2
C
yp
2B
C
yp 1
(M
C
yp 1)
(P
B
C 1)
yp
3A
1
C
yp
4A
C
yp 1
2D
22
N
A
D Cyp
PH
2
C E1
yp
re
d
U
dp .
gt
r2
A
ld
h
N
qo
1
p5
3
G
ad
d4
G
ad 5
d1
53
C
ox
-2
C
as
p3
Pc
n
C
yc a
lin
D
1
p2
1
Gene Expression Ratio
(relative to Gapdh)
Gene Expression Profile Obtained in Glitazone
-Treated Rat Primary Hepatocytes
*
Pioglitazone 100 uM
*
*
*
0
Apoptosis pathwaysmall cell lung cancer
Biotech J.com 2002
40-gene Multiplex Panel on GeXP
Additional References
Additional References
Additional References
Inspiration
Dedicated to improving
patient health and
reducing the cost of
care.