Post on 26-Mar-2021
Roche A
pplied Science
1
Accurate
Gene Expression A
nalysis with
High Flexibility: C
onceptsand D
evelopments
Dr. O
liver Geulen
Roche A
ppliedS
cienceR
esearch & D
evelopment, P
enzberg
Roche A
pplied Science
2
Gene Expression A
nalysisG
oals and R
equirem
ents
To quantitatively detect subtle changes in amounts of m
RN
A
against a complex background
To obtain reliable data that can be compared over a long period
of time or betw
een different experimental system
s
Prelim
inary Requirem
ents:A
ccurate and reproducible measurem
entsS
teady quantitative analysis of data
Roche A
pplied Science
3
RT-P
CR
Quantification
Influ
encin
g Factors
Roche A
pplied Science
4
LightCycler ®
System
s T
echnological (r)evolu
tions
Roche A
pplied Science
5
LightCycler ®
480 System
Sum
mary
New
ly developed, high-throughput system for real-tim
e PCR
.
Com
pact benchtopinstrum
ent for 96-and/or
384-microw
ell plates.
Optim
ized heating and cooling technology for increased speed and m
aximized
temperature uniform
ity.
Specifically developed optical system for m
aximized sensitivity
and the uniform collection of signals across the plate.
Roche A
pplied Science
6
Standard B
lock Cycler
Ram
ping
up T
emperatu
re
Roche A
pplied Science
7
LightCycler ®
480 Blockcycler
Therm
a-Base
TM
vacuum cham
ber saturated with w
orking fluidw
ick structure lining inside walls
hot location: evaporation into vacuum
cool location: condensation with heat release
Roche A
pplied Science
8
LightCycler ®
480 System
T
hermocycler
Therma-B
aseTM
Heatsink
MW
P M
ount
Heated
lids
IncludesThem
a-Base
TMfor
optimized
heatexchange.
Allow
sto finish a PC
R run
for96 w
ellsin <
1 hour and 384 w
ells in < 40 m
in.
Roche A
pplied Science
9
LightCycler ®
480 Instrument
OpticalSystem
Hom
ogeneous illumination and
imaging due to long object-
image distance.
Pinhole to mask lateral portions
of emitted light and focus on
central, perpendicular portions.
Large field lenseto efficiently
collect rays also from lateral
wells.
Filter Wheels
CC
D C
amera
MW
P 96/384
Roche A
pplied Science
10
LightCycler ®
480 Instrument
OpticalP
roperties
Lightsource: high intensityxenon
lamp
Lifetime: approx. 500-1000 h
450, 483, 523, 558, 615 nm
500, 533, 568, 610, 640, 670 nm
Filters forExcitation
Filters forEm
ission
Roche A
pplied Science
11
InternalReference
Dye
Internal reference dye is not required
due to accurate data generation (thermoblock)
data detection (optical system)
data analysis (software)
Roche A
pplied Science
12
LightCycler ®
480 System
Assay Form
ats, Dyes, an
d Application
483
450 (+/-15)
483 (+/-17.5)
523 (+/-10)
558 (+/-15)
558 (+/-15)
615 (+/-15)
483
483
Excitation(nm
)
Quantification
LightCycler ®
CY
AN
500FA
MVIC
/HEX
LightCycler ®
RED
610LightC
ycler ®R
ED 640
Cy5
500 (+/-10)
533 (+/-10)
568 (+/-10)
610 (+/-10)
640 (+/-10)
670 (+/-10)
Hydrolysis Probes
SNP A
nalysisFluorescein
533Sim
pleProbeProbes
Quantification
SNP A
nalysisFluo
-LightC
ycler ®R
ED 610
Fluo-
LightCycler ®
RED
640 Fluo
-C
y5
533/ 610533/ 640533/ 670
HybProbe
Probes
Qualitative D
etectionProduct -C
haracterizationQ
uantification
SYB
R G
reen I530
SYB
R G
reen l
Application
Dyes
Detection (nm
) A
ssay Format
Roche A
pplied Science
13
0.18010.1612
Standard deviation
65.456.85
Maxim
um
64.6756.14
Minim
um
64.8856.47
Average
Tm
(2)oC
T
m(1)
oC
LightCycler ®
480 Genotyping
Intra-In
strum
ent R
eproducibility
MD
R-1 C
3435T polymorphism
; SimpleProbe
Format.
Ninety-six replicates for each of the 3 different genotypes.
Roche A
pplied Science
14
LightCycler ®
480 qPC
R P
erformance
Intra-R
un
Reprodu
cibility
n.a.n.a.
1n.a.
n.a.1.00E
+010.1092
30.851.00E
+020.0921
27.311.00E
+030.0899
24.131.00E
+040.0315
20.851.00E
+050.0549
17.771.00E
+06SD
Mean
Copies
Viral target detected with H
ybProbeprobes
Roche A
pplied Science
15
Accuracy of LightC
ycler ®480 S
ystemD
iscrimin
ation of 500 an
d 1000 copies
7 replicates of human
genomic D
NA
distributed
over MW
P
Hydrolysis Probe Form
at
196 bpam
plicon50 000
25 000
10 000
1 000 500100
50
Roche A
pplied Science
16
Gene Expression A
nalysisG
oals and R
equirem
ents
To quantitatively detect subtle changes in amounts of m
RN
A
against a complex background
To obtain reliable data that can be compared over a long period
of time or betw
een different experimental system
s
Prelim
inary Requirem
ents:A
ccurate and reproducible measurem
entsS
teady quantitative analysis of data
Roche A
pplied Science
17
Typesof Q
uantification
Roche A
pplied Science
18
Relative Q
uantificationR
elative Ratio
Am
ount of target RN
A in a sam
pleA
mount of housekeeping R
NA
in a sample
For calculation:
N =
N0
x 2n
Nnum
berof am
plifiedm
oleculesN
0initialnum
berof m
oleculen
number
of amplification
cycles
Roche A
pplied Science
19
Monitoring of P
CR
Reactions
From B
lock Cyclers
to Real-T
ime P
CR
Displayed
amplification
curveis
influencedby
Detection
format
Fluorescencedye
Reaction
conditions, e.g. pH
Generation of C
p/Ctis
influencedby
algorithmused
Roche A
pplied Science
20
PC
R Efficiency
Am
plificationC
urve
vsStan
dards
Efficiencyis
a synonym for
qualityof P
CR
.
IsP
CR
efficiencyreflected
bystandard
curvesor
byindividualam
plificationcurves?
Roche A
pplied Science
21
Determ
ination of Am
plification EfficiencyA
mplification
Cu
rvevs
Standards
Fluorescence
Roche A
pplied Science
22
Calculation of P
CR
EfficiencyD
erivedfrom
Am
plificationC
urve
Phenom
enological descriptions of efficiency depending on individual am
plification curve of samples
so far no full automation possible
quality depending on users ability / algorithms used
so far only estimation of efficiency
Standardization critical
Roche A
pplied Science
23
Calculation of P
CR
EfficiencyD
erivedfrom
Standards
Efficiency based on dilution series
Prerequisite for Standards: EfficiencyStandard
= Efficiency
Sample
Statistical approach (amount of standards)
Laborious
Standardization
possible, butnotreflecting
thedifferences
of amplification
efficienciesof individual
samples
Reflects
individualPC
R´s
behaviour
Roche A
pplied Science
24
Linear vs. Non-Linear
PC
R Efficiency
Cu
rveFit D
epends
on D
ata
linear fitnon-linear
fit
Roche A
pplied Science
25
Relative Q
uantificationW
ithC
alibratorN
ormalization
A calibrator provides com
parison of many PC
R experim
ents (used as a “positive control”)
A calibrator corrects for differences in detection sensitivity
between target and reference genes
A calibrator does not correct for differences in PC
R
efficiency between the target and reference gene
Roche A
pplied Science
26
Relative Q
uantificationM
ethods(1)
Kn
own
as∆∆C
t Method
Calibrator N
ormalization w
ithout Efficiency Correction
This method assum
es that reference gene and target gene are am
plified with the sam
e efficiencyThis m
ethod assumes that the PC
R efficiency of both genes is 2
An efficiency correction w
ould significantly reduce calculation errors, because
Not every PC
R-System
is running with optim
al/identical PCR
efficiency (E =
2)N
ot every PCR
-System is even follow
ing a constant PCR
efficiency
Roche A
pplied Science
27
Relative Q
uantificationM
ethods(2)
Calibrator N
ormalization w
ith Efficiency Consideration
Relative R
atio = E
TC
pT(C
) -C
pT(S
)X
ER
CpR
(S) -
CpR
(C)
Uses the individual PC
R efficiency in the calculaton
Efficiency is generated via linear/polynomial standard curves
Roche A
pplied Science
28
Com
parison of Methods
Validity of C
alculated V
alues
Predefined GM
O standards of know
n concentrations (in %
), are analysedw
ith relative quantification with or w
ithout efficency
correctionSam
ple preparation greatly affects results
0.04%0.08%
0.1%0.28%
0.49%0.5%
0.45%0.73%
1.0%0.04%
0.08%0.1%
0.31%0.51%
0.5%0.67%
1.06%1.0%
Result
without
EfficiencyC
orrection(E=
2)R
esultw
ithEfficiency
Correction
GM
O
Content
Roche A
pplied Science
29
Standardization in G
ene Expression Su
mm
ary
Try to minim
ize technical variation
Use appropriate instrum
entation with low
variance and high reproducibility
Use highly accurate softw
are algorithms for qP
CR
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