Workshop :

Analisa Kuantitatif dengan Universal Probe Library Roche di real time PCR

Deka Lestario : Research Specialist

deka.lestario@roche.com /081316907019

Helen L. Utama : Application Specialist

helen_listiarini.utama@roche.com/ 0811 9191922

Run down
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12.00
12.15

Roche Introduction & pre test

presentasi teori dasar & basic real time PCR
Presentasi Analisa Quantitative & UPL
Diskusi
Persiapan
Running pcr / hands on / refreshing
Diskusi hasil
Post test and closing

Roche
A leading global healthcare company

Founded 1896 in Basel, Switzerland

Founding families still hold majority

stake

Employing around 80000 people

Currently active in 90 countries on all

continents

Clear focus on healthcare

Leadership in pharmaceuticals (#5)

leading supplier of medicines for
cancer and a market leader in virology

Leadership in in vitro diagnostics (# 1)

Unique innovation model

Worlds biggest biotech company*

More than 117 years of Roche

Historical timeline
Sales in 2008: 45617 million
CHF
50.000

40.000

Strategic Transition - focus

on Pharmaceuticals &
Diagnostics
Ventana
Chugai
Sale OTC
Disetronic
Boehringer
Mannheim

30.000

Genentech

Vitamins & Fine

Chemicals

Spin off
Fragrances & Flavours

Diagnostics

20.000

10.000

Personalized
Healthcare

Limited
Company
Founding

0
1896

1934

Fragrances &
Flavours

Internat.
expansion

1970

Sales

Diversification
1990

2000

2008

The Roche Global Group

Two divisions focused on high-value healthcare

Diagnostics
Roche
Applied
Science

Roche
Roche
Roche
Molecular Professional
Tissue
Diagnostics Diagnostics Diagnostics

Pharmaceuticals
Roche
Diabetes
Care

Roche
Pharma

Expertise in biology and technologies

Genentech

Chugai

Roche Diagnostics Overview

Operating five business areas in three markets
Life Science

In Vitro Diagnostics

In Vitro
Diagnostics

Researchers

Healthcare Professionals

Patients

Academia Pharma Biotech

Commercial labsHospitals Wards/Clinics Physicians

Applied Science

Patients

Consumers

Molecular Diagnostics
Professional Diagnostics
Tissue Diagnostics

Diabetes Care

Sample

Roche Applied Science

Solution Provider for Life Science Research

Cells

Organs

Organisms

Communities

Target

Subcellular

Detection

DNA, RNA

RT-PCR

Microarray

Sequencing

Cells

Tissues

Function

IHC/ISH

Cell Isolation
Genomics

Cytomics

ROCHE APPLIED SCIENCE

Reagents

Instruments

DNA / RNA Isolation

& PCR Family
Reagents
Light Cycler
Systems
Apoptosis, Cell Death
and Cell Proliferation Assays
Micro Array Systems
Nonradioactive In Situ
Hybridization
Automated for Purification

Genome
Sequencer

Transfection reagents

Tools for Mapping &

Cloning
Cell Anayzer
Array Services

The Roche LightCycler Story

14 Years of Continuous qPCR Innovation

LightCycler Nano
32 samples

LightCycler 96
96 samples

LightCycler 1536
1,536 samples
LightCycler 480
96 or 384 samples
LightCycler

Carousel

32 samples

2005

2009

2011

2012

1998
9

Pre Test

Nama
:
Departemen/Lab/ Insitusi:
Alamat Email / HP :
Research
:
1. Perbedaan antara realtime PCR dan PCR konven adalah :
a. siklus denaturasi, penempelan primer, pemanjangan
b. bisa qualitative PCR
c. bisa quantitative PCR
2. Jumlah siklus dimana sampel mulai terbaca diatas level ambang
batas fluorescence dikenal juga dengan sebutan
a. AFL
b. Cp/Ct
c. Tm
3. Panjang gelombang FAM dibawa di channel deteksi
a. 510
b. 560
c. 640

Polymerase Chain Reaction

Teori dan Pengertian Dasar

DNA
DNA

Chromosome

Gene

Materi genetik berupa Deoxyribonucleic Acid

(DNA)
Unit molekul yang mengkode informasi: Gen

Nucleus Chromosome

Kromosom merupakan unit penyimpan materi

genetik yang efisien. Manusia mempunyai 46
kromosom, berisi informasi genetik yang
lengkap

Materi genetik tsb terdapat dalam nukleus

dari suatu sel

Masing-masing sel akan berisi genome

manusia ( 3x109 DNA )

Struktur DNA

Double Helix
DNA Strand

Chromosome

DNA dan RNA

Perbedaan antara RNA dan DNA

Ribose

RNA

DNA

Ribose

Deoxyribose

Adenine (A)

Adenine (A)

Cytosine (C)

Cytosine (C)

Uracil (U)

Thymine (T)

Guanine (G)

Guanine (G)

No. of strands

Usually single

Double

Heat stable?

No

Yes

Sugar

Bases

Deoxyribose

DNA dan Basa Komplemen

Hydrogen Bonds
Cytosine
(C)
Cytosine
(C)
Adenine (A)
Adenine
(A)
Guanine (G)
Thymine
Thymine (T)
(T)
Guanine (G)
Guanine

Thymine (T)
Adenine (A)
Cytosine (C)

Deoxyribose
(Sugar molecule)

Phosphoric Acid
(Phosphate molecule)

DNA , RNA , Protein

Gene 2

DNA
molecule

Gene 1
Gene 3

DNA strand

A C C A A A C C G A G T

(template)

TRANSCRIPTION

mRNA

U G G U U U G G C U C A

Codon
TRANSLATION

Protein

Figure 17.4

Trp
Amino acid

Phe

Gly

Ser

Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin

Cummings

In eukaryotes
RNA transcripts are modified before
becoming true mRNA

In prokaryotes
Transcription and translation occur together

Nuclear
envelope

DNA
TRANSCRIPTION
Pre-mRNA

TRANSCRIPTION

RNA PROCESSING

DNA

mRNA

mRNA
Ribosome
TRANSLATION
Ribosome
TRANSLATION

Polypeptide
Polypeptide

Eukaryotic cell. The nucleus provides a separate

compartment for transcription. The original RNA
transcript, called pre-mRNA, is processed in various
ways before leaving the nucleus as mRNA.

Prokaryotic cell. In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated
without additional processing.

Kode Genetik
Empat Basa

Tiga Basa (triplet)

TRANSCRIPTION

DNA
mRNA
Ribosome

ATG CAG TTT TGA

TRANSLATION
Polypeptide

Satu kodon mendefinisasikan satu asam

amino (20 AA)

Amino
acids

Polypeptide

ATG

Methionine
43 kombinasi membentuk 64 kodon
DNA

tRNA with
amino acid

Ribosome attached

Gene
Exon 1 Intron Exon 2
Transcription
RNA processing

Intron Exon 3

Gly
tRNA

Translation
Domain 3
Domain 2
Domain 1
Polypeptide

A A A
U G G U U U G G C

Codons

5
mRNA

Anticodon
3

Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin

Cummings

Concept 17.6: Comparing gene expression in prokaryotes and eukaryotes

reveals key differences
Prokaryotic cells lack a nuclear envelope
Allowing translation to begin while transcription is still in progress
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase

Direction of

0.25 m

transcription

DNA

Polyribosome
Polypeptide
(amino end)
Ribosome

Figure 17.22

mRNA (5 end)

Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin

Cummings

A summary of
transcription and
translation in a
eukaryotic cell

DNA

TRANSCRIPTION
1 RNA is transcribed

from a DNA template.

3

RNA

RNA

transcript
2

polymerase
Exon

RNA PROCESSING
In eukaryotes, the

RNA transcript (pre-

RNA transcript

mRNA) is spliced
and

(pre-mRNA)
Intron
Aminoacyl-tRNA

modified to produce
mRNA, which moves
from the nucleus to
the
cytoplasm.
CYTOPLASM

synthetase

NUCLEUS
Amino

FORMATION OF

acid
tRNA

INITIATION COMPLEX
3

After leaving the

AMINO ACID ACTIVATION

4

nucleus, mRNA attaches

Each amino acid

attaches to its proper tRNA

to the ribosome.
mRNA

with the help of a specific

Growing
polypeptide

enzyme and ATP.

Activated

amino acid
Ribosomal

subunits

TRANSLATION
5

A A A
UGGUUUA UG

Codon

Figure 17.26

Ribosome

A succession of tRNAs

add their amino acids to

Anticodon

the polypeptide chain

as the mRNA is moved
through the ribosome
one codon at a time.
(When completed, the
polypeptide is released
from the ribosome.)

Protein

Creatine: Struktur
Rambut

Myosine: Kontraksi Otot-otot

Hemoglobin: Transport Oksigen

dalam Darah
Insulin: Degradasi Gula
Lipase : Memotong Lemak
Antibodies untuk Sistem Imun
(Kekebalan Tubuh)

Teknologi PCR

PCR = Polymerase Chain Reaction

Enzyme based site directed amplification
of nucleic acids

Polymerase: nama enzim utk polimerisasi

Chain reaction: reaksi berantai (amplifikasi)

Dikembangkan oleh Kary Mullis (USA) pada

1985
Nobel-price 1993 (en.wikipedia.org)

Benefits
Sample Uji

Mesin realtime pcr

Isolasi DNA /
RNA

Mastermix

Elektroforesis

- Pembuatan agar,
- Pewarnaan EtBR,
- proses
dokumentasi

Geldoc

Hasil

Deteksi Produk PCR:

Elektroforesis Gel agarosa Elisa : Microwell Plate Realtime PCR

Visualisasi Primer-Dimer

GeI electrophoresis

LightCycler Melting Curve Analysis

Realtime PCR Workflow

10
min

Pre treatment
Sample

45 min
Ekstraksi
DNA
( manual /
automatic
)

30 min
PCR-set
up

60 min

30 min

Proses PCR

Analisis
dan
deteksi

Tahapan Proses PCR

Pre PCR :
Preparasi reagensia
Preparasi spesimen: isolasi/ purifikasi DNA/RNA

PCR: proses amplifikasi

Sample ( DNA /RNA ) + Taq DNA Polymerase + dNTP + Primer ( Probe )
Denaturasi (pemisahan rantai DNA)
Annealing (penempelan primer)
Extension (pemanjangan oleh enzim)
Post PCR:
Deteksi/Analisa Hasil PCR

Preparasi spesimen:
Isolasi/Ekstraksi/Purifikasi Asam Nukleat
dengan Metode Phenol

Isolasi
DNA

Isolasi RNA

Preparasi spesimen:
Isolasi/Ekstraksi/Purifikasi Asam Nukleat
dengan Metode Column

Berikatan dengan glass

fleece
Garam kaotropik

Wash and spin

Pelarutan asam nukleat
Analisa selanjutnya

Preparasi
Reagensia:
Komponen Master
Mix

PCR Buffer,
2+
Mg

Customer spesifik:
1. DNA/RNA Template
2. Sekuen primer

and
OR

Mn

Taq DNA
Polymerase

2+

and

dCTP
dGTP
dUTP

Primer

dATP
Deoxynucleotides
(dNTPs)
Probe

Status Sample :
1. Sample : Unknown
2. Kontrol Positif
3. Kontrol Negatif : NTC
4. Reference ( Housekeeping )
5. Standard curve

1. Principles of PCR

Initial
Denaturation 92 98C

THE CYCLE
Denaturation
92 98C

Elongation
68 72C
Annealing
50 68C

Cooling
4- 8C

Reverse
Transcription

cDNA

RNA Virus

PCR Review
cDNA

Target
Denaturation

Reverse Transcription

and Polymerase Chain

Reaction

Primer
Annealing

Target Amplification
Primers
Polymerase & dNTPs

Extension

PCR Amplification

No. Amplicon Cycles

No. of Cycles Copies of Target
1

16

32

20

1,048,576

30

1,073,741,824

Teori - PCR

N = N0 x
N:
N0:
n:

n
2

jumlah molekul teramplifikasi

jumlah molekul awal
jumlah siklus amplifikasi

Kuantifikasi PCR :
Aspek Teori dan Praktek

N = N0 x 2n
log-phase-PCR

Theory
N = N0 x (Econst)n

Real

N = N0 x (Evar)n

N: number of amplified molecules

molecules
n: number of amplification cycles

end-point-PCR

N0: initial number of

E: amplification efficiency

PCR and the Problem of Quantification

background phase

log-phase analysis end-point analysis

N
N
n

high concentration /
high efficiency
high concentration /
low efficiency
low concentration /
high efficiency
: number of amplified molecules
: number of amplification cycles

Crossing Point / CP
6.00

Norm Fluorescence

5.00
4.00

Ct = 33.2

3.00
2.00
1.00

AFL

0.00
0
-1.00

10

20

30

CP

40

50

60

Cycles

Data fluoresensi dikumpulkan pada setiap siklus.

Nilai Critical Threshold (Ct) (ambang batas kritis), diartikan sebagai
jumlah siklus dimana sampel mulai terbaca diatas arbitrary
fluorescence level (AFL), menunjukkan awal mulainya fase
pertumbuhan exponensial.
Ct dikenal juga dengan sebutan Crossing Point/ CP.

Primer and Probes

Pelacak

Pewarna DNA

Fluorescence Spectroscopy

Senyawa Fluorescent menyerap

cahaya pada panjang gelombang
tertentu
Absorbance spectrum

Cahaya yang diserap diemisikan

Fluorescence
spectrum

Sumber cahaya putih melalui filter

untuk memilih warna spesifik yang
mengoptimasi absorpsi fluorescent
Excitation filter

Warna spesifik pada spektrum

fluorescen dibaca dengan detektor
Emission filter

Absorbance
spectrum

Fluorescence
spectrum

Format Detektor dalam Realtime PCR

Intercalating dye

Sybr green I, Eva green

Resolight Dye/ HRM

Non Intercalating dye

Hyb Probe

Taqman Probe

Format SYBR Green I / HRM

Ketika SYBR Green I berikatan dengan primer dsDNA, akan terjadi

peningkatan fluoresensi

Selama tahapan PCR yang berbeda, intensitas dari sinyal fluoresensi

akan berbeda, tergantung dari jumlah dsDNA yang ada

Format Hybridization Probe / HybProbe

Hybridization probe merubah fluoresensi pada saat hibridisasi dengan
fluorescene resonance energy transfer (FRET)
2 probe oligonukleotida sekuen spesifik dilabel dengan pewarna yang
berbeda (donor & aseptor) dan ditambahkan kedalam master mix
Dalam analisa HybProbe adanya produk amplifikasi spesifik dapat dibaca
secara kuantitatif berdasarkan peningkatan fluoresensi

Fluorescei
n

LC Red

Format Hybridization Probe / HybProbe

Spesifik karena 2 probe dihibridisasi dengan target dengan cara yang
sangat sekuen spesifik
Primer-dimer tidak terdeteksi karena probe sekuen spesifik tidak
mengenalinya
Dapat untuk aplikasi deteksi mutasi, analisis SNP, genotyping SNP , qPCR
dan multiplex tes

Format Hydrolysis Probes

Dikenal juga dengan nama Taqman Probe
Menggunakan aktivitas exonuclease 5 dari DNA Polymerase
Sebuah probe terdiri dari 2 label, fluorescence reporter dan fluorescene
quencher, yang sangat dekat satu sama lain. Ketika probe masih utuh,
quencher menahan sinyal fluoresensi reporter

reporte
r

quencher

Format Hydrolysis Probes

Pada proses PCR, aktivitas exonuclease 5 dari DNA Pol memotong
hidrolisis probe dan memisahkan reporter dan quencher
Sinyal fluoresensi reporter tidak lagi tertahan dan dapat memancarkan
sinyal fluoresensi

Peningkatan sinyal fluoresensi dari reporter berbanding langsung dengan

akumulasi produk PCR

Format Simple Probes

Simple Probe adalah bentuk sederhana dari hybridization probe dan hanya
menggunakan 1 probe saja
Ketika terjadi hibridisasi akan memancarkan sinyal fluoresensi yang lebih besar

Inovasi Roche : Format Simple Probes

Perubahan sinyal fluoresensi tergantung dari status hibridasi dari probe,
semakin stabil hibridisasinya semakin tinggi temperatur melting
Untuk aplikasi SNP genotyping dan deteksi mutasi

LightCycler Assay Formats

SYBR Green I

Elongation Phase

Hybridization Probe Format

Hydrolysis Probe Format

Annealing Phase

Elongation Phase

Simple Probe Format

Hal yang diperhatikan dalam memilih realtime PCR

Ramping rate ( kecepatan naik turunnya suhu}

Akurasi suhu
Homogenisitas Suhu
Kapasitas sample ( upgradable 96 to 384 ? )
Easy Handling instrument ( no need calibration if we remove the machine )
HRM, High Resolution Melting ( akurasi suhu tinggi)
Software easy to use , easy programming , easy analysis in :
1. Absolut & relative Quantification
2. Melting curve analysis
3. Realtime ready * ( no need optimalization in designing new primer / probe )
( Universal Probe Library & Realtime Ready Panel )

Hal penting lainnya :

- Garansi oleh Principal , bukan oleh Agen
- Harga mastermix dan reagen yang terjangkau
- Training oleh professional / dedicated specialist

LightCycler 480 System

Components

Instrument

Reagent Kits

Block Kit

Multiwell Plates

Software Modules

Prevalidated Probes

LightCycler 480 System

96 384 Switch
Interchangeable thermal
block cycler
Do-it-yourself, fast
Loading help for
convenience
No service engineer
required

No re-calibration
necessary
Instrument automatically
detects and identifies
block

LightCycler 480 Thermal Block Cycler

Speed and Accuracy
Homogenous
temperature
distribution over
the plate
Fast PCR runs:
96 wells in < 1
hour 384 wells in
< 40 min
Therma-Base
for optimized heat
equalization

The Roche LightCycler 96 System

Newest Addition to the LightCycler Family

I.

Accurate Data Generation & Data Capture

II.

Intuitive Interface and Smart Analysis Tools

III.

Key Features & Reliability

LightCycler 96 System

56

Analysis Tools
Intuitive Instrument Interface
Sensitive state-of-the-art 10
Touch Screen and onboard
computer for complete
standalone functionality
First time non experienced
qPCR users found it very
intuitive and extremely easy to
navigate and to operate, within
a few clicks
5 predefined programs
Fully generate, execute and
monitor experiments

LightCycler 96 Touch Screen Interface

57

Aplikasi Realtime PCR

Powerful Applications

59

Quantification Principles

Overview

Real-Time PCR Quantification Principles

Absolute Quantification :

Relative Quantification :

Target and Reference as the same

gene

Target and Reference used two genes

External
Standards
(monocolor)

External
Standards
(without calibrator)

External
Standards with
Internal Control
(dual-color)

( gene of interest & Housekeeping gene )

CalibratorNormalized
Method

60

Absolute Quantification

Relative Quantification :
Menggunakan 2 gen :
1. Gene of interest ( GOI )
2. Housekeeping gene / Reference

Housekeeping genes code for proteins that are

essential to cellular function

Assumptions:
Housekeeping genes are expressed constitutively and their expression levels are identical
in all samples to be analyzed
62
Expression level is not regulated in the experimental system
normal vs. tumor tissue, or
treated vs. untreated cells

Housekeeping Genes
Gene

Genomic structure / pseudogenes

Regulation e.g.

-actin

multigene family; > 20 genes; 1 active locus

: hormones of tyroid gland

20 pseudogenes

: stomach tumor

g-actin

multigene family; pseudogenes

GAPDH

multigene family; 10-30 genes; > 200 in mouse

: lung, pancreatic, colon cancer

mostly pseudogenes

: insulin, EGF

5.8S,18S, 28S RNA

pseudogenes

2-microglobulin

no pseudogenes

: Non-Hodgkin lymhoma
abnormal expression in tumors

G6PDH

no pseudogenes

: kidney, stomach tumor

: hormones, oxidant stress,
growth factors

PBGD

no pseudogenes

aldolase

pseudogenes

HPRT

pseudogenes

U3, U8, ...

Pseudogenes

ornithin

: tumors

decarboxylase
...

63

Relative Quantification
1. Metode CT
CT COX2 ( Target )

CT GAPDH ( reference )

Calibrator ( Kontrol Sehat )

15.0

16.5

Test ( Pasien kanker )

12.0

15.9

1.

Nomalisasi CT gene target ke reference

2 ( Ct GAPDH Ct COX2 ) = relative espresi
Calibrator
:
2 ( C16.5-15.0 ) = 2.8
Test ( cancer ) :
2 ( 15.9-12.0 ) = 14.9
2. Nomalisasi rasio ekspresi
Ekspresi kontrol
: 2.8/2.8
Ekspresi sel target : 14.9 / 2.8 = 5.3
Artinya COX2 diekspresikan 5.3 kali lipat pada pasien kanker
dibandingkan orang sehat.

Relative Quantification
2. Metode Livak
CT COX2 ( Target )

CT GAPDH ( reference )

Calibrator ( Kontrol Sehat )

15.0

16.5

Test ( Pasien kanker )

12.0

15.9

1.
Nomalisasi CT gene target ke reference
CT ( calibrator) = CT ( target, cal ) - CT (reference, cal )
= CT (control ) = 15.0 16.5 = -1.5
CT ( test ) = CT ( target, tes ) - CT (reference, cal )
= CT (control ) = 12.0 15.9 = -3.9
2. Nomalisasi CT test ke CT calibrator
CT = CT ( test ) - CT (cal ) = -3.9 ( -1.5) = -2.4

3. Hitung rasio level ekspresi

2 CT = 2 (-2.4) = 5.3
Artinya COX2 diekspresikan 5.3 kali lipat pada pasien kanker dibandingkan orang sehat.

Relative Quantification
3. Metode Kurva Standar Reference
Unknown Sample

Fluorescence

Crossing Point

Target: COX2

Fluorescence

Standards : G6pdh with human template

Cycles

Log Concentration

Cycles

Concentration of Target
Result

Cycles

Reference : GAPD

Standards : G6pdh with human template

Log Concentration

Fluorescence

Fluorescence

Crossing Point

RASIO = Concentration of Reference

Cycles
66

Prinsip Kuantifikasi Relatif dengan Standard External

Unknown Sample

log (F2/F1)

Target

cycles

Standard Curve

Crossing Point (Cycles)

log (F2/F1)

cycles

Konsentrasi

Cp/Ct

Sample zdhhcpre

2100. ??

28.

Sample zdhhcpost

50 ??

35

Sample HKgapdpre

1078 ??

32

Sample HKgapdpost

1350 ??

31

StandarmurA 1

1000

25

StandarmurA 2

10

30

Kontrol negatif

Kontrol positif murA

15

26

Concentration of Target
Result
RASIO =
log (copy number)

Concentration of HKgapd

Determination of Efficiency of Target and Reference

Cover at least 3-5 orders of

magnitude in the range of the
samples to be analyzed
Use at minimum of 4-5 dilution
steps (e.g., 1:10 dilutions)
Use 3-6 replicates each,
for a valid statistical basis

68

Various PCR Efficiencies between Target

and Reference
Cp

1. Efficiency = 2 (CT method)

Log Conc.
Cp

2. Efficiency adjusted (linear)

Log Conc.
Cp

Log Conc.

3. Efficiency adjusted (non-linear)

Cp

Log Conc.

69

Relative Quantification Methods

Effect of Efficiency Differences

70

Error Generation

Dependence of Analysis Error on PCR Efficency and Cycle Number

PCR Efficiency

2.00
1.97
1.95
1.90
1.80
1.70
1.60

Detection Cycle (n)

10
15
20
16%
25%
35%
29%
46%
66%
67% 116%
179%
187% 385%
722%
408% 1045% 2480%
830% 2740% 8570%

25
46%
88%
260%
1290%
5710%
26400%

30
57%
113%
365%
2260%
13000%
80700%

35
70%
142%
500%
3900%
29500%
246400%

Error Calculation (2n/En-1) x 100

71

Relative Quantification Methods (2)

- Method

An Efficiency consideration significantly reduces

calculation errors due to differences in amplification
of target and reference genes.

The Roche Applied Science

-method

normalized relative ratio

=
ET CpT (C) - CpT (S) X ER CpR (S) - CpR (C)
Calibrator normalization with efficiency consideration uses the individual
PCR efficiency in the calculation
72

Innovasi Roche :
Relative quantification using standard curves

Calibrator normalized relative quantification (Ct Method)

Calibrator normalized relative quantification using standard curves for
efficiency correction.

Relative Quantification Experiments (2)

Monocolor/Dual Color

requires color compensation

depending on dye combination

74

Other Applications

Protocol Melting curve

Melting curve program

Melting curve with : SYBR Green I

Quantification

Melting Curve
Template: human genomic DNA; Target: -Globin; Detection Format: SYBR Green I

Melting curve with : Simple Probe

Melting curve with : Hyb Probe

LightCycler Genotyping: Example II

SNP with Three Different Alleles (e.g. Apolipoprotein B)
Template:
Plasmid DNAs

Single Color:

LC RED 640

80

LightCycler Genotyping: Example III

Two Loci/Amplicons, One Color

(e.g. Hemochromatosis)

Bernard et al. (1998), AJP 153,1055

81

LightCycler Genotyping: Example IV

2 Amplicons, 4 SNPs, 2 Colors

82

Aplikasi PCR / real time PCR

Penyakit Infeksi / Non infeksi

Dengue
HIV RNA genotyping
Salmonella thyposa
Enterovirus
HBV DNA - cccDNA
Rotavirus
HCV RNA micro RNA
H5N1
MTB DNA
H1N1
Legionella
CMV DNA
Polio Virus
HPV DNA genotipe 16 & 18
Helicobacter pylori
Herpes Virus

Toxoplasmosis
Fecal Microbiota
Sepsis (gram +/ garm -/ Candida)
EBV

Colorectal cancer :
k-ras
Anti-EGFR
Leukemia:
Bcr-abl
Thalasemia :
Alfa & beta-globin
Breast cancer :
P53
Brca1 & brca2
PTEN
Progesteron reseptor
Estrogen reseptor

Application : Forensics

Biological Evidence
~ paternity/maternity testing

~ linkage of suspects to crime scenes

Identification of Individuals
~ missing persons and casualties

* Polri
* RSCM

* Eijkman Lab Genneka

Applications : Zoonosis Study

Balai Teknik Kesehatan dan Lingkungan
Balai Pengendalian Penyakit Bersumber Binatang
- Leptospira, PES, Chikungunya, Dengue, Rabies, H5N1

Aplication : Food, Drug and Vaccine Safety

* BPOM, LPPOM MUI, BIOFARMA, Food Factory ( Nestle, Indolakto )
STREAK OUT

Day
1

Day

ON ALOA +
PALCAM
37C FOR 24 - 48h

25 g/ml TEST SAMPLE

IN 225 ml 1/2 STRENGTH FRASER
30C FOR 24h
0.1 ml IN 10 ml FRASER

35 OR 37C FOR 48h

3-4
STREAK OUT
ON ALOA + PALCAM

Day
3-6

Day

4-7

37C FOR 24 - 48h

All colonies showing a blue-green color with opaque halo / grey-green color with a black zone are
presumptive L. monocytogenes colonies (typical colonies) and counted as such. Suspect colonies must be
confirmed.

BIOCHEMICAL CONFIRMATION: GRAM (+), CATALASE (+), MOTILITY (+), CAMP

(+), HAEMOLYSIS (+), GLUCOSE (+), RHAMNOSE (+), XYLOSE (-)

Applications :
Balai Karantina
- Animal : H5N1

- Fish : Koi Herpes Virus,

WSSV, TSV
- Plants : Pantoea ananas &
Microcyclus ulei

Applications :Plantations
(1) LEAF / TISSUE
SAMPLING

London Sumatera : Sawit

Asian AGRI : Sawit
Astra Agro : Kertas
SMART : Sawit
East West Seed : Jagung dll
Balit Sayur Lembang
Puslit Buah Tropika
Balit Tanaman Serat
Puslit Kakao
Balitbiogen
IRRI
Balit Jeruk dan Subtropika
Balit Tanaman Hias Ciherang
Balit Padi Bogor
Balai Mutu Benih Horti
Balit OPT Horti & Pangan
Pusat Kajian Buah IPB

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Applications : Vaccine Industry

* BIOFARMA, VAKSINDO, SANBE, Biotek Indonesia, MEDION,
Caprifarmindo

Realtime Ready
Universal Probe Library : Taqman
LightMix : Hyb Taq
LightSnip : Hyb Probe
Configurator

Realtime Ready :

Universal Probe Library

- Format Taqman Probe

- Sudah optimum ( suhu annealing 60 )
- RUO
- Open system to many Platform ( ABI, Rotorgene, Biorad )
- Open to any mastermix taqman

Probe Konvensional

Universal Probe Library Roche

Mencari jurnal dan Blast data ke NCBI

sendiri
Butuh Primer design software

Software flexible dan user friendly

Software open dan bisa diakses oleh
siapa saja

Belum optimum, karena beda mesin,

beda sumber sample, beda mastemix
beda pewarna DNA

Optimum untuk realtime Roche & open

ke realtime non Roche, open ke semua
tipe mastermix

Untuk menjalankan lebih dari 1 probe

harus menyamakan annealing

165 tabung UPL sudah optimum di

annealing yang sama yakni 60

Untuk ekspresi gen dengan relative quant

jika membutuhkan multiplexing yakni
pada sample dicek rasio gen target dan
gen housekeeping bersamaan harus
dalam 1 tabung.

Tersedia multiplexing analisa utk human

dan mouse.
Untuk UPL sudah dengan Taqman Probe
, memakai pewarna FAM sementara
HouseKeeping gene memakai
Yellow555

Waktu penelitian yang pendek

Time saving dan cost effective

Multiplexing dengan Housekeeping GeneAssay

Cost of Reagents :
1. RNA Isolation kit
2. Transcriptor cDNA sintesis kit
3. mastermix ( Hyb probe / Taqman / Sybrgreen )
4. Primer / Probe ( UPL )
5. Housekeeping gene ( * relative Quant )
6. Standar Curve
7. Positive control

8. template standard : plasmid , human genomic DNA

RT-PCR Quantification: Influencing Factors

Sample

DNA/RNA

Sample

Nucleic Acid

Reverse

Preparation

Isolation

Transcription

Preparation
Method

Isolation
Method

Stability of NA

Purity

Storage

Variability of
Isolation

Product

cDNA

Amplification

Efficiency

Efficiency

Enzyme
Variability

Enzyme
Variability

Detection

Detection
Method
Linearity of
Assay

Storage

106

Realtime Ready :
LightSniP
- Format Hybprobe dan Simple Probe
- Sudah optimum
- RUO
- Closed system to Lightcycler Platform

One -Hemoglobin SNP

Realtime Ready :

LightMix
- Format Hybprobe dan Taqman Probe
- Sudah optimum
- RUO dan beberapa ada yang sudah IVD
- Closed system to Lightcycler Platform

Contoh Lightmix ( CE dan RUO )

40-0095-16

05947146001

hu

MTHFR C677T RUO

RU

40-0099-32

05947219001

hu

PAI-4G/5G to be replaced by 40-099-64

RU

40-0099-64

06896359001

hu

PAI-4G/5G CE

CE

40-0129-64

06896367001

hu

MTHFR C677T CE

CE

40-0135-16

05947189001

hu

PML-RAR

RU

40-0137-16

05945305001

hu

G6PDH (reference gene)

RU

40-0196-16

05945682001

hu

AML1-ETO

RU

40-0229-16

05945593001

hu

inv 16

RU

40-0269-32

05945810001

hu

MTHFR A1298C

RU

40-0269-64

06896383001

hu

MTHFR A1298C CE

CE

Lightmix (1)

Lightmix (2)

Lightmix (3)

Lightmix (4)

Post Test

Nama
Departemen/Lab/ Insitusi:
Alamat Email / HP :
Research
:

1. Pembuatan kurva standar pada relative quantifikasi memerlukan perhatian pada

a. Tm
b. Cp
c. Efisiensi

2. Format probe yang memberikan sinyal saat annealing adalah :

a. Taqman Probe
b. Hyb probe
c. Simple Probe

3. Untuk penelitian ekspresi gen dengan realtime pCR dengan UPL terdapat fitur, kecuali :
a. intron spanning assay
b. housekeeping gene assay
c. Melting curve analysis

Terima kasih

UPL Wet Workshop

February 2015

Housekeeping
Gene

Template: Human gDNA

Primer and probe : UPL
Human G6PD

Std. Curve

Template: Human gDNA

Primer and probe: UPL
Human G6PD

Serial Dilution for Standard Curve

Stock concentration: 200 ng/ul

102 (200 ng/ ul)

101 (20 ng/ul):

10 ul human gDNA [102]+ 90 ul ddH2O

100 (2 ng/ul):

10 ul human gDNA [101] + 90 ul ddH2O

Formula Master mix:

FastStart Taqman Probe Master
Component

Conc.

Volume

Final Conc.

Primer Mix (UPL Human

G6PD)

20 uM

0.5 ul

500 nM

Probe (UPL Human G6PD)

10 uM

0.5 ul

250 nM

FS Taqman Probe Master

2x

10 ul

1x

Water

4 ul

Total Volume
Template

15 ul
5 ul

Final Volume

20 ul

PCR Protocol

Laboratory Set-up

Doing now what patients need next