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

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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

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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

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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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