PPT Workshop Realtime Ready_Cost

Deka Lestario : Research Specialist

[email protected] /081316907019

Helen L. Utama : Application Specialist

[email protected]/ 0811 9191922

Workshop :

Analisa Kuantitatif dengan Universal Probe Library Roche di real time PCR

Run down

08.30 08.45 Roche Introduction & pre test

08.45 09.45 presentasi teori dasar & basic real time PCR

09.45 10.15 Presentasi Analisa Quantitative & UPL

10.15 10.30 Diskusi

10.30 10.45 Persiapan

10.45 11.30 Running pcr / hands on / refreshing

11.30 12.00 Diskusi hasil

12.00 12.15 Post test and closing

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Cytomics Genomics

RT-PCR

Sa

mp

le

Ta

rge

t D

ete

cti

on

Subcellular Communities Organisms Organs Cells

Cells DNA, RNA Tissues

IHC/ISH

Cell Isolation

Microarray Sequencing Function

Reagents

Apoptosis, Cell Death

and Cell Proliferation Assays

Nonradioactive In Situ

Hybridization

Transfection reagents

DNA / RNA Isolation

& PCR Family

Reagents

Tools for Mapping &

Cloning

Instruments

Micro Array Systems

Genome

Sequencer

Automated for Purification

Light Cycler

Systems

Cell Anayzer

ROCHE APPLIED SCIENCE

Array Services

1998

2005 2009

2011 2012

9

The Roche LightCycler Story 14 Years of Continuous qPCR Innovation

LightCycler 1536

1,536 samples

LightCycler Nano

32 samples

LightCycler 480

96 or 384 samples

LightCycler Carousel

32 samples

LightCycler 96

96 samples

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

Materi genetik berupa Deoxyribonucleic Acid (DNA)

Unit molekul yang mengkode informasi: Gen

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

Gene

Nucleus Chromosome

Chromosome

DNA

Masing-masing sel akan berisi genome manusia ( 3x109 DNA )

Double Helix

DNA Strand

Chromosome

Struktur DNA

DNA dan RNA

RNA DNA

Sugar Ribose Deoxyribose

Adenine (A) Adenine (A)

Bases Cytosine (C) Cytosine (C)

Uracil (U) Thymine (T)

Guanine (G) Guanine (G)

No. of strands Usually single Double

Heat stable? No Yes

Perbedaan antara RNA dan DNA Ribose Deoxyribose

DNA dan Basa Komplemen

Cytosine (C)

Adenine (A)

Thymine (T)

Guanine (G) Guanine (G)

Guanine (G)

Thymine (T)

Adenine (A)

Adenine (A)

Thymine (T)

Cytosine

(C)

Cytosine (C)

Hydrogen Bonds

Deoxyribose

(Sugar molecule) Phosphoric Acid

(Phosphate molecule)

DNA , RNA , Protein

Figure 17.4

DNA

molecule

Gene 1

Gene 2

Gene 3

DNA strand

(template)

TRANSCRIPTION

mRNA

Protein

TRANSLATION

Amino acid

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

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

Trp Phe Gly Ser

Codon

3 5

3 5

In prokaryotes

Transcription and translation occur together

Prokaryotic cell. In a cell lacking a nucleus, mRNA

produced by transcription is immediately translated

without additional processing.

TRANSLATION

TRANSCRIPTION DNA

mRNA

Ribosome

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.

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclear

envelope

In eukaryotes

RNA transcripts are modified before

becoming true mRNA

Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin

Cummings

Kode Genetik

Empat Basa A C T G

Tiga Basa (triplet)

ATG CAG TTT TGA

Satu kodon mendefinisasikan satu asam

amino (20 AA)

ATG Methionine

43 kombinasi membentuk 64 kodon

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

Polypeptide

Amino

acids

tRNA with

amino acid

attached Ribosome

tRNA

Anticodon

mRNA

Gly

A A A

U G G U U U G G C

Codons 5 3

Gene DNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

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

Figure 17.22

DNA

Polyribosome

mRNA

Direction of

transcription 0.25 m RNA

polymerase

Polyribosome

Ribosome

DNA

mRNA (5 end)

RNA polymerase

Polypeptide

(amino end)


Cummings

A summary of transcription and translation in a eukaryotic cell

Figure 17.26


Cummings

TRANSCRIPTION

RNA is transcribed

from a DNA template.

DNA

RNA

polymerase

RNA

transcript

RNA PROCESSING In eukaryotes, the

RNA transcript (pre-

mRNA) is spliced

and

modified to produce

mRNA, which moves

from the nucleus to

the

cytoplasm.

Exon

RNA transcript

(pre-mRNA)

Intron

NUCLEUS

FORMATION OF

INITIATION COMPLEX

After leaving the

nucleus, mRNA attaches

to the ribosome.

CYTOPLASM

mRNA Growing

polypeptide

Ribosomal

subunits

Aminoacyl-tRNA

synthetase

Amino

acid

tRNA AMINO ACID ACTIVATION

Each amino acid

attaches to its proper tRNA

with the help of a specific

enzyme and ATP. Activated

amino acid

TRANSLATION

A succession of tRNAs

add their amino acids to

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.)

Anticodon A A A

U G G U U U A U G

E A

Ribosome

1

5

5

3

Codon

2

3 4

5

Protein

Creatine: Struktur Rambut

Myosine: Kontraksi Otot-otot

Hemoglobin: Transport Oksigen

dalam Darah

Lipase : Memotong Lemak

Insulin: Degradasi Gula

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

Isolasi DNA /

RNA

Mesin realtime pcr

Elektroforesis Geldoc Hasil

Mastermix

Sample Uji

- Pembuatan agar,

- Pewarnaan EtBR,

- proses

dokumentasi

dengan geldoc

Deteksi Produk PCR:

Elektroforesis Gel agarosa Elisa : Microwell Plate Realtime PCR

Visualisasi Primer-Dimer

GeI electrophoresis LightCycler Melting Curve Analysis

10

min 45 min 30 min 30 min

60 min

PCR-set

up

Proses PCR Ekstraksi

DNA

( manual /

automatic

)

Pre treatment

Sample

Analisis

dan

deteksi

Realtime PCR Workflow

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,

Mg2+

Mn2+

dCTP

dGTP

dUTP

dATP

Taq DNA

Polymerase

Primer

and

OR

and

Deoxynucleotides

(dNTPs)

Customer spesifik:

1. DNA/RNA Template

2. Sekuen primer

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

Cooling

4- 8C

Denaturation

92 98C

Elongation

68 72C

Annealing

50 68C

THE CYCLE

Target

Denaturation

Primer

Annealing

Primers

Extension

Polymerase & dNTPs

PCR Review

Reverse Transcription

and Polymerase Chain

Reaction

RNA Virus

cDNA

Target Amplification

cDNA

Reverse

Transcription

PCR Amplification

No. of Cycles No. Amplicon Cycles

Copies of Target

1 2

2 4

3 8

4 16

5 32

20 1,048,576

30 1,073,741,824

Teori - PCR

N = N0 x 2n

N: jumlah molekul teramplifikasi

N0: jumlah molekul awal

n: jumlah siklus amplifikasi

Kuantifikasi PCR :

Aspek Teori dan Praktek

N: number of amplified molecules N0: initial number of

molecules

n: number of amplification cycles E: amplification efficiency

Real

Theory

end-point-PCR

log-phase-PCR

N = N0 x (Econst)n

N = N0 x 2n

N = N0 x (Evar)n

PCR and the Problem of Quantification

high concentration / high efficiency

high concentration / low efficiency

low concentration / high efficiency

N : number of amplified molecules

n : number of amplification cycles

log-phase analysis

N

n

end-point analysis background phase

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 10 20 30 40 50 60

Cycles

Norm

Flu

ore

scence

Ct = 33.2

AFL

Crossing Point / CP

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.

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

Sybr green I, Eva green

Resolight Dye/ HRM

Non Intercalating dye

Hyb Probe

Taqman Probe

Intercalating dye

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 Hybridization Probe Format Hydrolysis Probe Format

Elongation Phase 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 Prevalidated Probes Software Modules

Multiwell Plates Block Kit


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

56


Analysis Tools Intuitive Instrument Interface

LightCycler 96 Touch Screen Interface

57

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

Aplikasi Realtime PCR

59

Powerful Applications

60

Real-Time PCR Quantification Principles

Absolute Quantification :

Target and Reference as the same

gene

Relative Quantification :

Target and Reference used two genes

( gene of interest & Housekeeping gene )

External

Standards

(monocolor)

External

Standards

(without calibrator)

Calibrator-

Normalized

Method

External

Standards with

Internal Control

(dual-color)

Quantification Principles

Overview

Absolute Quantification

62

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

Expression level is not regulated in the experimental system

normal vs. tumor tissue, or

treated vs. untreated cells

63

Housekeeping Genes

-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

...

Gene Genomic structure / pseudogenes Regulation e.g.

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.


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.

66

Flu

ore

scen

ce

Cycles

Reference : GAPD

Flu

ore

scen

ce

Cycles

Target: COX2 Unknown Sample

Result

RASIO =

Concentration of Target

Concentration of Reference

Cycles

Flu

ore

scen

ce

Standards : G6pdh with human template

Cycles

Flu

ore

scen

ce

Cro

ssin

g P

oin

t

Log Concentration

Cro

ssin

g P

oin

t

Log Concentration

Standards : G6pdh with human template


3. Metode Kurva Standar Reference

Prinsip Kuantifikasi Relatif dengan Standard External

lo

g (

F2/F

1)

Target

Unknown Sample

cycles

cycles

log

(F2/F

1)

Standard Curve

Cro

ssin

g P

oin

t (C

ycle

s)

log (copy number)

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 0 0

Kontrol positif murA 15 26

Result

RASIO =

Concentration of Target

Concentration of HKgapd

68

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

69

Various PCR Efficiencies between Target

and Reference

1. Efficiency = 2 (CT method)

2. Efficiency adjusted (linear)

3. Efficiency adjusted (non-linear)

Cp

Log Conc.

Cp

Log Conc.

Cp

Log Conc.

Cp

Log Conc.

70

Relative Quantification Methods

Effect of Efficiency Differences

71

Error Generation

Detection Cycle (n)

10 15 20 25 30 35

PC

R E

fficie

ncy

2.00 - - - - - -

1.97 16% 25% 35% 46% 57% 70%

1.95 29% 46% 66% 88% 113% 142%

1.90 67% 116% 179% 260% 365% 500%

1.80 187% 385% 722% 1290% 2260% 3900%

1.70 408% 1045% 2480% 5710% 13000% 29500%

1.60 830% 2740% 8570% 26400% 80700% 246400%

Error Calculation (2n/E

n-1) x 100

Dependence of Analysis Error on PCR Efficency and Cycle Number

72

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

Innovasi Roche :

Relative quantification using standard curves

Calibrator normalized relative quantification (Ct Method)

Calibrator normalized relative quantification using standard curves for efficiency correction.

74

Relative Quantification Experiments (2)

Monocolor/Dual Color

requires color compensation

depending on dye combination

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

80

LightCycler Genotyping: Example II

SNP with Three Different Alleles (e.g. Apolipoprotein B)

Template: Plasmid DNAs

Single Color:

LC RED 640

Melting curve with : Hyb Probe

81

LightCycler Genotyping: Example III

Two Loci/Amplicons, One Color (e.g. Hemochromatosis)

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

82

LightCycler Genotyping: Example IV

2 Amplicons, 4 SNPs, 2 Colors

Aplikasi PCR / real time PCR

Penyakit Infeksi / Non infeksi

HIV RNA genotyping

HBV DNA - cccDNA

HCV RNA micro RNA

MTB DNA

CMV DNA

HPV DNA genotipe 16 & 18

Herpes Virus

Toxoplasmosis

Fecal Microbiota

Sepsis (gram +/ garm -/ Candida)

EBV

Dengue

Salmonella thyposa

Enterovirus

Rotavirus

H5N1

H1N1

Legionella

Polio Virus

Helicobacter pylori

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

Day

1

Day

3-6

25 g/ml TEST SAMPLE

IN 225 ml 1/2 STRENGTH FRASER

30C FOR 24h

STREAK OUT

ON ALOA + PALCAM

37C FOR 24 - 48h

Day

3-4

Aplication : Food, Drug and Vaccine Safety

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

Day

4-7

0.1 ml IN 10 ml FRASER

35 OR 37C FOR 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.

STREAK OUT

ON ALOA +

PALCAM

37C FOR 24 - 48h

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

(1) LEAF / TISSUE

SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Applications :Plantations

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

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

106

RT-PCR Quantification: Influencing Factors

DNA/RNA

Isolation

Method

Purity

Variability of

Isolation

Storage

cDNA

Efficiency

Enzyme

Variability

Product

Detection

Method

Linearity of

Assay

Sample

Preparation

Method

Stability of NA

Storage

Sample

Preparation

Nucleic Acid

Isolation

Reverse

Transcription Amplification Detection

Efficiency

Enzyme

Variability

Realtime Ready :

LightSniP

- Format Hybprobe dan Simple Probe

- Sudah optimum

- RUO

- Closed system to Lightcycler Platform

One -Hemoglobin SNP

LightMix

- Format Hybprobe dan Taqman Probe

- Sudah optimum

- RUO dan beberapa ada yang sudah IVD

- Closed system to Lightcycler Platform

Realtime Ready :

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

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

Nama :

Departemen/Lab/ Insitusi:

Alamat Email / HP :

Research :

Terima kasih

UPL Wet Workshop

February 2015

Template: Human gDNA

Primer and probe : UPL Human G6PD

House-keeping

Gene

Template: Human gDNA

Primer and probe: UPL Human G6PD

Std. Curve

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 15 ul

Template 5 ul

Final Volume 20 ul

PCR Protocol

Laboratory Set-up

Doing now what patients need next

PPT Workshop Realtime Ready_Cost

Documents

Transcript of PPT Workshop Realtime Ready_Cost