Download - 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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Founded 1896 in Basel, Switzerland
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stake
Employing around 80000 people
Currently active in 90 countries on all
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Leadership in pharmaceuticals (#5)
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Unique innovation model
Worlds biggest biotech company*
Roche
A leading global healthcare company
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More than 117 years of Roche Historical timeline
0
10.000
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30.000
40.000
50.000
1896 1934 1970 1990 2000 2008
Sales
Founding Internat.
expansion
Fragrances &
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Diagnostics
Genentech
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Fragrances & Flavours
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Chemicals
Diversification
Strategic Transition - focus
on Pharmaceuticals &
Diagnostics
Sales in 2008: 45617 million
CHF
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Limited
Company
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Pharmaceuticals Diagnostics
Roche
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Science
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Diagnostics
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Molecular
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Tissue
Diagnostics
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Expertise in biology and technologies
Roche
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Care
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Pharma
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Roche Diagnostics Overview Operating five business areas in three markets
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Researchers
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Patients
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In Vitro
Diagnostics
Tissue Diagnostics
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Roche Applied Science Solution Provider for Life Science Research
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
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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
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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
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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
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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
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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)
Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin
Cummings
-
A summary of transcription and translation in a eukaryotic cell
Figure 17.26
Copyright 2008 Pearson Education Inc., publishing as PearsonBenjamin
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
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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)
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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)
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Benefits
Isolasi DNA /
RNA
Mesin realtime pcr
Elektroforesis Geldoc Hasil
Mastermix
Sample Uji
- Pembuatan agar,
- Pewarnaan EtBR,
- proses
dokumentasi
dengan geldoc
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Deteksi Produk PCR:
Elektroforesis Gel agarosa Elisa : Microwell Plate Realtime PCR
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Visualisasi Primer-Dimer
GeI electrophoresis LightCycler Melting Curve Analysis
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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
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Preparasi spesimen:
Isolasi/Ekstraksi/Purifikasi Asam Nukleat
dengan Metode Phenol
Isolasi
DNA
Isolasi RNA
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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
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1. Principles of PCR
Initial
Denaturation 92 -
98C
Cooling
4- 8C
Denaturation
92 98C
Elongation
68 72C
Annealing
50 68C
THE CYCLE
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Target
Denaturation
Primer
Annealing
Primers
Extension
Polymerase & dNTPs
PCR Review
Reverse Transcription
and Polymerase Chain
Reaction
RNA Virus
cDNA
Target Amplification
cDNA
Reverse
Transcription
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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
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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
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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
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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
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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
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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
-
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
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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
LightCycler 96 System
-
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
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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.
-
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.
-
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
Relative Quantification
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