overview agilent 2100 bioanalyzer solutions lab on a ... · celular y proteínas (western)...
TRANSCRIPT
Page 1
Overview Agilent
Lab on a
ChipTechnology
2100 Bioanalyzer Solutions
Cordoba 8 de Abril
Agilent 15 Marzo 2010
Page 2
Slab gel analysis a bottleneck in
Protein & DNA / RNA analysis
• Manual process
• Difficult to automate
• Slow
• Not accurate enough
• Bad reproducibility
• No direct comparison
• No digital data
Lab-on-a-Chip technology integrates the analysis typically done on
Slab gel and UV-photometers to provide unattended runs and digital
Qualitative and quantitative Data in 30-40 minutes.
Agilent 15 Marzo 2010
Page 3
The Agilent 2100 bioanalyzer offers
On a single Platform perform:
• Sizing, Quantitation and Purity of Proteins (5 -230 kDa)
• Sizing, Quantitation and Purity of DNA fragments (25 – 12000 bp)
• Integrity check, Separation and Quantitation of RNAs
• Cell Fluorescence Assays with stained cells (Apoptosis, Transfection, Expression, )
Improved quality of slab-gel-type analysis
through on-Chip electrophoresis…
Easy to use analytical device that performs
separation and quantitation of RNA, DNA and
Proteins based on electrophoresis within a
disposable glass chip.
1. Load sample 2. Run analysis 3. Analyze data
…reducing the experiment to:
Agilent 15 Marzo 2010
Page 4
Chip Architecture
• Chip accommodates sample wells, gel
and conditioning/destaining wells, and
a well for a standard (ladder)
Gel wells
Ladder well
Sample wells
Ladder
well
Gel wells
Separation channel and point of detection
Conditioning
well
• 16 pin-electrodes in the electrode
cartridge (standard equipment) are
arranged such that they fit in the wells on
the chip
Agilent 15 Marzo 2010Page 5
The LabChip® Approach - Simplified Model
+
_+
-
Agilent 15 Marzo 2010Page 6
Principle of Electrodriven FlowUsed for molecular assays (analysis of DNA, RNA and proteins)
On-chip gel electrophoresis
The sample
moves electro-
driven from the
sample well
through the
micro-channels
The sample
is electro-
kinetically
injected into
the separa-
tion channel
Sample
components
are electro-
phoretically
separated
Components are
detected by their
fluorescence and
translated into
gel-like images
(bands) and
electrophe-
rograms (peaks)
The micro-
channels of the
glass chip are
filled with a
sieving polymer
and fluorescent
dye
Agilent 15 Marzo 2010Page 7
Lab-on-a-Chip - Principle of Injection & Separation
3
2
4
Direction of electrodriven movement of liquids and molecules within liquids
1
Separation channel Inje
ction
ch
an
ne
l
Agilent 15 Marzo 20010Page 8
2100 Bioanalyzer LabChip Kits
What you need to know
• Chips are disposable, they are used once and then thrown away
• Even if all wells on a chip are not been used the remaining wells
cannot be used at a later time
• Some of the reagents in the LabChip kits are thermally unstable at
room temperature so the kits have to be shipped cooled (on ice) and
stored in a refrigerator
• The LabChip kits have a limited shelf life
– Shelf life is 12 months from manufacture date
– We guarantee a minimum of 4 months shelf-life when received by
the customer
• Reagent kits (cooled) are available for customers who lose/waste their
reagents or have reagents that exceeded their shelf-life
Page 9
2100 expert SW Version B.02.07
Third SW Generation• Includes all the new Series II bioanalyzer kits
• Supporting new High Sens DNA and Protein
• Patended RIN (RNA Integrity Number)
• Color coded Result Flagging
• Allows compliance also on e-bioanalyzer (G2939AA)
• Improved comparison context (up to 48 samples per file)
• Improved manual integration
• Customizable result tables for printing and reports
• Not compatible with A-type Instruments
Agilent 15 Marzo 2010
Agilent 15 Marzo 2010
Page 10
Gel view and electropherogram view
→time/size
↑ fluorescence intensity/
quantity
Agilent 15 Marzo 2010
Page 11
0. preparatory transformations
• Noise reduction
(Savitzky-Golay filtering
• Baseline correction
• Spike rejection
Baseline correction disabledBaseline correction enabled
Agilent 15 Marzo 2010
Page 12
1. Peak recognition
table of
fluorescence
intensities
table of
peaks
Result table in Expert
Electropherogram in Expert
Agilent 15 Marzo 2010
Page 13
2. Alignment to markers
Lower marker
Upper marker
Analysis turned off –
Unaligned data
Analysis turned on –
Aligned data
Agilent 15 Marzo 2010
Page 14
3. Sizing
size:
ladder
Agilent 15 Marzo 2010
Page 15
4. Quantification (DNA/Protein samples)
peak area
concentration
upper marker peak area
(known concentration)
assay-specific calibration curve
(standard areas, see assay properties)
Ladder
Agilent 15 Marzo 2010
Page 16
Tree view for navigation between
samples and files
Context
menu bar
Customizable
gel-like image
(change order)
Customizable result table
(change order and add
additional columns)
Single gel lane
for selected
E-gram
Task bar with context
sensitive icons for
different actions
Tabs for different
data displays
Access to
setpoint
explorer
Page 17
Agilent Web pages with 2100 content
www.opengenomics.com
www.agilent.com/chem/labonachip
tAgilent 15 Marzo 2010
Page 18
Aplicaciones con tecnología
microfluidica en ARN, ADN, Contaje
Celular y Proteínas (Western)
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Lab-on-a-Chip
Control Activo de Fluidos (Microfluídica)
Volumen de Muestra 1 -4 µl
10 -12 muestras dependiendo de
aplicación.
Separación, ticción, detección de
muestras.
Resultados en 5-30 minutos disponibles
No necesidad de un sistema desecho.
No contaminación cruzada.
Page 20
Current Kits & Assays (including anything required for
analysis)
Protein Assays:• Sizing
• Quantitation
• cell lysates, column fractions,
purified proteins, antibodies etc.
• 10 samples in 40 min.
P230 P80
DNA Assays:• Sizing
• Quantitation
• PCR products, digests,
larger DNA fragments
• 12 samples in 30 min.
DNA1000 DNA7500 DNA12000
Cell Assays:
• Analysis of 6 samples
• Two color detection
• Analysis of protein expression
in cells
Flow Cytometry
RNA Assays:
• Quantitation (Sizing in Small RNA)
• total RNA, mRNA
• purity & integrity
determination
• 10 samples in 30 min.
6000 Nano 6000 Pico Small RNA
HSP 230
High Sensitivity DNA Cell Check Out
Agilent 15 Marzo 2010
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RNA Applications
RNA QA/QC
for
Microarrays
Gene
Expression
Genomic
DNA
ContaminationRNA QA/QC
for qPCR
RNA QA/QC
for mPCR
smallRNA
QA/QC
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Analytical
Specification total RNA mRNA
Qualitative Range 5-500ng/ul 25-250ng/ul
Quantitative Range 25-500ng/ul 25-250ng/ul
Reproducibility of
Quantitation 10% CV 10% CV
Maximum Sample
Buffer Strength 10 mM Tris-EDTA 10 mM Tris-EDTA
Physical
Specification
Analysis Time 30 minutes 30 minutes
Number of
Samples 12 samples/chip 12 samples/chip
Sample Volume 1ul 1ul
Assay Kit Stability 4 months at 4 C 4 months at 4 C
Plataforma para RNA
Analytical
Specification total RNA mRNA
Qualitative Range 50-5000 pg/µl 250-5000 pg/µl
Maximum Sample
Buffer Strength 50 mM Tris or NaCl 50 mM Tris or NaCl
Physical
Specification
Analysis Time 30 minutes 30 minutes
Number of
Samples 11 samples/chip 11 samples/chip
Sample Volume 1 µl 1 µl
Assay Kit Stability 4 months at 4 C 4 months at 4 C
RNA 6000 Nano LabChip kit RNA 6000 Pico LabChip kit
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total RNA
Determina integridad y calidad de ARNt.
Determinación de concentración de ARN.
Identificar picos ribosomales.
Calcula el ratio de picos ribosomales (18S/28S or 16S/23S)
RNA integrity number (RIN)
mRNA
Determina integridad y calidad de muestras de mRNA
Determinación de concentración de RNAm
Calcula % ribosomal RNA en muestras de ARNm
Caracteristicas del RNA 6000 chip
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RNA 6000 Nano LabChip kit Analisis de Total RNA Integrity
total RNA alta calidad
total RNA parcialmente degradado
18
S
28
S
Flu
ore
sce
nce
Time (seconds)
0
1
2
3
4
5
6
7
8
9
10
11
19 24 29 34 39 44 49 54 59 64 69
marker18S
28S
Flu
ore
scence
Time (seconds)
0
1
2
3
4
5
6
7
8
9
10
11
19 24 29 34 39 44 49 54 59 64 69
marker
28S18S Típico primer QC despues de
obtener el ARN antes de su
aplicación para microarrays o
real-time PCR
2100 bioanalyzer: electropherogram
2100 bioanalyzer: single lane gel-like image
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Gel Chip Comparación
Falso Negativo
Data kindly provided by Gene Logic Inc.
Falso Positivo
Data kindly provided by Gene Logic Inc.
1.09 ratio 28S/18S
0.90 ratio28S/18S
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Gene Expression ; RNA QC como Rutina
Empezar de
nuevo con
aislamiento
Células / Cultivos
Aislamiento ARN
Total RNA
RIN
RNA QC via Agilent 2100 bioanalyzer
RIN sobre el limite
Continuar con los posteriores experimentos (Microarray, real-time PCR, etc.)
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108 muestras en 3 diluciones
CV RIN: 3 %
CV ribosomal ratio: 22 %
RIN – COMPARATIVA DE MUESTRAS
18
S
28
S
Flu
ore
sce
nce
0.0
2.5
5.0
7.5
10.0
12.5
18S
28S
Flu
ore
scence
Time (seconds)
0
10
20
30
40
50
60
70
80
19 24 29 34 39 44 49 54 59 64 69
18S
28S
Flu
ore
scence
0
5
10
15
20
25 ng/µl: RIN 8
100 ng/µl: RIN 8
500 ng/µl: RIN 8
Identico RIN en la misma
muestra con varias diluciones.
REPRODUCIBILIDAD!!
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RIN Aplicaciones – Medidas de la Integridad del ARN
ARN Intacto: RIN 10
ARN parcialmente degradado:
RIN 5
Fuertemente degradado: RIN 3
18
S
28
S
Flu
ore
scence
0
1
2
3
4
5
6
7
8
9
18
S
28
S
Flu
ore
scence
0
5
10
15
20
25
30
35
40
45
Flu
ore
scence
Time (seconds)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
19 24 29 34 39 44 49 54 59 64 69
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cRNA f ragment at i on
Cy3/ Cy5 Label i ng
Tot al RNA QC
mRNA QC
Ar r ay exper i ment
Aplicaciones en microarrays
Dat a eval uat i on
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Month ##, 200XPage 30
18S 28S
Fluoresce
nceTime (seconds)
0.0
2.5
5.0
7.5
10.0
12.5
16 21 26 31 36 41 46 51 56 61 66 71 76 81
18S 28S
Fluoresce
nce
Time (seconds)
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
21 26 31 36 41 46 51 56 61 66 71 76 81
ARNr contaminación: 19,5%
ARNr contaminación: 1.7 %
Mo
use
kid
ney
I m
RN
A
Mo
use
kid
ney
II
mR
NA
Ra
t b
rain
mR
NA
Bo
vin
e k
idn
ey m
RN
A
Mo
use
tes
tis
mR
NA
Mo
use
liv
er m
RN
A
RN
A 6
00
0 l
ad
der
Contaminación de ARN Ribosomal en muestras de
ARNm
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Month ##, 200XPage 31
Analisis de Small RNA (usando RNA 6000 Assay)
Small RNA fracciones:
< 200 nts ejm; miRNA,
siRNA, snRNA, tRNA,
5S RNA
Permite
discriminar entre
diferentes
perfiles.
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Small ARN especificaciones
Rango Analitico 6 -150 nt
Sensibilidad 50 pg/µl
Rango Quantitativo 50 pg/µl – 2000 pg/µl
Reproducibilidad 25 % CV
Max cantidad ARN total 100 ng/µl total RNA
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Sm
all
RN
A R
eg
ion
Nuevo Small RNA versus RNA 6000 nano
miR
NA
Reg
ion
Lo
wer
Mark
er
Small RNA Region
tRN
A
18s
28s
RIN: 8.1
Lo
wer
Mark
er
RNA 6000Nano kit
Small RNA Kit
RNA 6000Nano
Rango tamaño 25-6000nt
Resultados: Integridad, cantidad de ARNt,
gDNA contaminacion
NEW! Small RNA
Rango tamaño: 6-150nt
Resultados: Cantidad de miARN Ratio y otros
Small RNA
5s
5.8
s
Muestras miRNA en musc.esqueletico
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DNA Applications
mtDNA
Screening
qPCR
validation,
impurity check
mPCR
validation,
impurity check
Restriction
Digest
Analysis
Gene
Expression
Oncology
Food
Analysis
Clinical
Research
Forensic
Testing
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Aplicaciones plataforma DNA
Pureza de los productos de PCR
Amplificaciones multiples por PCR
Gene expression análisis via RT-PCR (validación target)
GMO testing
Detección de Patogenos (Veterinaria, hospitales, medio
ambiente)
Aplicaciones en Genotipado
• Duplicaciones/ delecciones
• Frequencia alélica
• Sub-tipado de bacterias
• Forense
Cancer diagnóstico
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marker
marker
25 bp
473, 500 bp
100, 105 bp
300, 315 bp
900, 1000 bp
2100 bioanalyzer
25 bp
473, 500 bp
100, 105 bp
300, 315 bp
900, 1000 bp
2 % agarosa gel (Ticción BrEt)
Resultados - Bioanalizador vs Agarosa Gel
DNA QC in NGS – Reference Publication
High Sensitivity DNA Kit
May 2009
DNA QC in Next-Gen Sequencing -
20 pg/µl
2 pg/µl
Electropherogram overlay of 4 DNA fragment peaks: 20 pg/µl, 10 pg/µl, 5 pg/µl, 2 pg/µl
High Sensitivity DNA Kit – Detection limit
Page 40
PCR Product Analysis using the
DNA 7500 or DNA 1000 Kit
Page 41
The PCR Reaction
1. Denature dsDNA
2. Anneal primer
5’ 3’3’ 5’
3. Extension
Repeat steps 1-3
Amplified product
Exponential DNA amplification
1983, Kary Banks Mullis, noble prize 1993
Page 42
Determination of PCR Product Impurity
1 2 3 4 5
20001200
200
800
400
100
1 2 3 4 5
2000
1200
200
800
400
100
Agilent 2100 Bioanalyzer agarose gel
300 bp
3000 bp
DN
A 7
500
Page 43
quantitative data from Agilent 2100 Bioanalyzer
Sample c (DNA) main peak
300 bp PCR 41.4 ng/ul 40.7 ng/ul
300 bp PCR 1:4 9.6 ng/ul 9.6 ng/ul
Sample c (DNA) main peak
3000 bp PCR 61.9 ng/ul 40.7 ng/ul
3000 bp PCR 1:4 14.8 ng/ul 9.8 ng/ul
1* 2 3 4*
Fluoresc
ence
Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
300 bp PCR 300 bp PCR 1:4 dil.
Impurity level: < 2 %
1* 2 3 4 5 6 7*
Fluoresc
ence
Time (seconds)
0
100
200
300
400
500
25 30 35 40 45 50 55 60 65 70 75 80 85 90
3000 bp PCR 3000 bp PCR 1:4 dil.
Impurity level : >30 %
300 bp
3000 bp
Determination of PCR Product ImpurityD
NA
7500
Page 44
1* 2 3 4 5 6 7 8*
Fluore
scence
Time (seconds)
0
10
20
30
40
50
60
70
80
90
35 40 45 50 55 60 65 70 75 80 85 90 95
mINF1G-TNF : 351 bp
mINF1G-IL1 : 294 bp
mINF1G-IL6 : 453 bp
mINF1G-GMC : 200 bp
mINF1G-TGF : 249 bp
mINF1G-GAP : 532 bp.
Data kindly provided by Maxim Biotech
Multiplex RT-PCR of 6 mouse inflammatory genesD
NA
1000
Page 45
Restriction Fragment Analysis
Page 46
Mutation Detection by RFLP
Wild type Mutant
Point mutationRestriction fragment cleavage site
Restriction
enzyme
digestion
+
2 fragments 1 fragment
Page 47
Detection of Single Base Mutations (1)in Exons 7 and 8 of the Human p53 Gene by RFLP Mapping using the DNA 7500 kit
Amplify exons 7 and 8 (resulting products:
618 bp fragment and 200 bp fragment)
Digest with Hpa II
Analyze using Agilent 2100 bioanalyzer
and 4-20 % acrylamide gel
exon 7 exon 8
83 bp, 91 bp,
168 bp, 276 bp
91 bp, 109 bp
In each example one of the restriction
sites can be deleted by a point mutation
DN
A 7
500
Page 48
Detección de Mutaciones puntuales (2)
wt 106
200 bp109 bp
91 bp
91 bp91+83 bp
168 bp
wt 59
276 bp276 bp
251 bp
1* 2 3 4 5*0
5
10
15
20
25
30
Fluo
resc
ence
Time (seconds)
30 35 40 45 50 55 60 65 70 75
p53Exon 7 wt/HpaII p53Exon 7 clone 59/HpaII
Lower
Marker
Upper
Marker267/268 bp
251 bp
166 bp
84/85 bp
1* 2 3 4*
0
5
10
15
20
25
Fluo
resc
ence
25 30 35 40 45 50 55 60 65
p53Exon 8 wt/HpaII p53Exon 8 clone 106/HpaII
Lower
Marker
Upper
Marker
Time (seconds)
208 bp
111 bp
90 bp
1HORA VS 90 SEC
Page 49
Peak Nummer Actual size (bp) Chip result (bp) % Error
2 119 110 -7.63 149 149 04 641 643 0.35 815 818 0.46 1195 1230 2.97 2058 1960 -4.88 2800 2622 -6.49 3588 3426 -4.5
10 4182 4191 0.211 4845 4745 -2.112 6297 5850 -7.113 9228 8594 -6.9
1*
2 3 4 5 6 7 8 9 10
11
12
13
14
*
Flu
ore
sce
nce
Time (seconds)
0
25
50
75
100
125
150
25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Adenovirus 2/Dra I
upper
marker
lower
marker
agarose gel Agilent 2100 bioanalyzer data
Peaks not detected
on agarose gel
2058
9228
2800
35884182
4845
6297
1195
815
641
149
119
DNA Analysis - linear dynamic rangeD
NA
12000
Page 50
Detección GMO: Determinación de porcentaje
de GM en Soja
Data kindly provided by CCFRA
Soya lectin
gene target
EPSPS gen: Especifico para Roundup
Ready GM soja (Monsanto)
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Diagnostico de Tumores
Data kindly provided by Adnagen
1. Inyeccion de celulas cancerigenas en sangre de
donante.
2. Enriquecimiento con AdneGen Cancer Select kit
(antibody based immunomagnetic enrichment.)
3. Multiples amplificaciones con AdnaGen
CancerDetect kit
4. Deteccion con Agilent 2100 Bioanalyzer and
DNA 1000 LabChip kit
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Cell Applications
Protein
Expression
Monitoring
Intracellular
Transfection
Efficiency
Monitoring
Apoptosis
Detection
Gene
Silencing
Protein
Expression
Monitoring
Cell Surface
Antibody
Staining
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Principales aplicaciones
Apoptosis:
Annexin V Detection of phosphatidylserine on the cell surface
Caspase-3 Detection of activated caspase-3 in the cytoplasm
Eficiencia de la transfección :
GFP: Detection of GFP-transfected cells
Antibody staining: Detection of transfected cells expressing the encoded protein
Monitorización de Expresión de proteinas :
Extracellular and Intracellular Antibody staining for detection of protein expressed on the cell surface, in the cytoplasm, or in the nucleus
Silenciamiento de genes
: Optimization of siRNA transfection procedure
Verify silencing by cellular protein expression measurement
Correlation of siRNA uptake and gene knockdown
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Flow Cytometry on a Chip
- Detección de 2 colores - 3 Tipos of Eventos
Blue/red cellsRed cells Blue cells
Dot plot view for easy
data evaluation
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:
Preparación de muestra:
adherent
suspension(trypsinize) & harvest
by centrifugation,
wash
wash twice
Resuspend cells in
cell buffer (LabChip Kit)
Load on chip
Add staining reagent
and incubate
Data analysis
to result
Customer Agilent
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2100 Bioanalyzer
Red detection channel:
• 620-645 nm excitation with Laser (Maximum 630 nm)
• 674-696 nm detection range (Maximum 680 nm)
Blue detection channel:
• 458-482 nm excitation with LED (Maximum 470 nm)
• 510-540 nm detection range (Maximum 525 nm)
Flow Cytometry on a Chip - Optics & Detection
Standard Flow Cytometers have 3-4 fluorescence detection channels:
FL1: excitation 488 nm, detection 530 nm
FL2: excitation 488 nm, detection 585 nm
FL3: excitation 488 nm, detection 661 nm
FL4: excitation 635 nm, detection 670 nm
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Applications: Protein Expression AnalysisGFP Transfection Efficiency Control
Disclaimer. Neither Agilent nor Caliper makes any representation that use of EGFP and
practice of the methods described in this publication is licensed or
otherwise authorized under any third party patents, including patents owned
or exclusively licensed by Aurora Biosciences or Amersham Biosciences.
CHO-K1 cells were transfected with
EGFP DNA and Lipofectamine.
GFP transfected cells
Mock transfected cells
0.1 %
56.6 %
Control
EGFP
transfected
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GFP On-Chip Staining - Histogram Quality
2100 bioanalyzer
Flow
Cytometer
¿Qué es la Proteómica?
Page 60
-Proteina + Genoma…………..Proteoma(1994 Marck
Wilkins)
Proteoma
Dotación completa de proteínas, incluyendo las modificaciones hechas a un conjunto particular de proteínas, producidas por un organismo o sistema
Proteómica
Estructura
Función
- 25.000-30.000 genes……………………………………500.000-1.000.000 proteínas!!
Page 61
El estudio y comparación sistemáticos
del proteoma en diferentes situaciones
metabólicas y/o patológicas permite
identificar aquellas proteínas cuya
presencia, ausencia o alteración se
correlaciona con determinados
estadios fisiológicos.
BIOMARCADORES
Diagnosis
+
Evolución de
enfermedades
Page 62
Las principales áreas de estudio de la proteómica son:
1-Identificación de proteínas y caracterización de sus
modificaciones postraduccionales
2-Proteómica de "expresión diferencial“
3-Estudio de las interacciones proteína-proteína
Page 63
1-Identificación de proteínas y caracterización de sus modificaciones
postraducionales:
Enorme complejidad(gran número).
-Técnicas más comunes:
A.Electroforesis monodimensional
SDS-PAGE…1ºDesnaturalizar 2ºCargar 3ºMigrar por peso molecular
B.Electroforesis bidimensional
2D-PAGE…Separación por PI + SDS-PAGE……Proteoma complejo
C.Cromatografía Líquida:
-Por hidrofobicidad; Columnas de Fase reversa.
-Por carga eléctrica: Columnas de intercambio iónico
-Por tamaño: Exclusión molecular
Page 64
OFFGEL Bioanalyzer
Page 65
¿Estas trabajando con muestras peptidicas o proteicas
complejas?
¿Estas interesado en fraccionar grandes cantidades de
estas muestras?
¿Estas interesado en obtener mejor sensibilidad en tu LC-
MS por técnicas de prefraccionamiento?
¿Estas haciendo digestiones de proteinas in-gel?
¿Estas buscando variaciones en carga de la purificación de
proteínas?
Page 66
- Separación de proteínas y péptidos de acuerdo a sus puntos isoeléctricos.
- Utiliza un gradiente de pH inmovilizado sobre un gel (IPG)
- Se aplica un potencial, las proteínas migran a través del gel hasta que alcanzan un pH = pI.
Fundamento del OFFGEL
Punto Isoeléctrico: pH al cual la carga neta de la proteína es cero (se calcula en función del número de cadenas laterales básicas y ácidas)
Page 67
Aplicación:
Protein Enrichment by OFFGEL Electrophoresis
bLG
bLG
3 x
OFFGEL load OFFGEL Fraction pH 4.8
1 % bLG
0.1 % bLG
0.01 % bLG
no bLG
1 % bLG
0.1 % bLG
0.01 % bLG
no bLG
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 69
Protein Applications
Antibody
QA/QC
Protein
Expression
Protein
Purification
Food
Analysis
Protein
QA/QC
Compliant
Protein &
Antibody
QA/QC
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 70
Aplicaciones del Protein LabChip
Células lisadas• Identificación sobre proteinas expresadas
• Comparación de diferentes formas de expresión
Fracciones de columna• Monitorización de aislamiento de proteinas y
purificación.
• Comprobación de fracciones(impurezas)
Purificar proteinas• monitorizar impurezas
• QC de anticuerpos monoclonales y policlonales
Month ##, 200X
Protein LabChip Kits – Specifications (Series II)• Analisis automatizado de 10 muestras de proteinas en menos de 30 minutos
• Dos aplicaciones para diferentes tamaños
– Protein 80: 5 a 80 kDa
– Protein 230: 14 a 230 kDa
-Protein250: 10-250kDa
• Resolución del 10% sobre el rango de tamaño.
• Rango linear dinámico grande:
(e.g. from 15 - 2000 ng/ l CA-II in PBS)
• Sensivilidad equivalente a tinción no-coloidal con Coomassie (R-250)
• Cuantificación relativa y absoluta
• Compatible con variedad de tampones.
Staining, Destaining and DetectionTinción, Desteñido y Detección
proteina
micelas
desteñir
SDS + dye
detección
low background good signal
to noise ratio SDS conc.
below CMC
If no dilution was done the
micelles would result in high
background and low
sensitivity
detection
X
Page 73
Page 74
Expresión de proteínas
Page 75
Protein 230 kit
Purificación de proteínas
Page 76
Se obtienen resultado de tamaño, pureza y concentración
En un solo experimento, en 45 minutos y con 4µl de muestra
Protein 230 kit
Estudio cinético de la eliminación del HIS-tag
Page 77
Análisis de la capacidad de las columnas
Page 78
Protein 230 kit
Análisis de anticuerpos
Page 79
High sensitive
Protein 250 kit
Glicosilación de proteínas
Page 80
Protein 230 kit
Control de calidad de Anticuerpos
Page 81
Protein 230 kit
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 82
Quality Control of Antibodies
90 kDa 160 kDa
intact
antibody
16% half
antibody
Determine the half antibody content in IgG
preparations
Antibody analysis under reducing and non-reducing
conditions
Ab reduced
Ab non-
reduced
light chain
heavy
chain
intact
antibody
light + heavy
chain
Absolute Quantitation of IgG samples
Análisis de proteínas en leche
Page 83
Protein 80 kit
Immunoprecipitation/Western Blot:
IP/HSP-250
Bioanalyzer
High sensitive
Protein 250 kit
Geles en 2D: Combinación de Offgel y
Bioanalizador
Page 85
High sensitive
Protein 250 kit
E. coli lysate
(50 g)
OFFGEL Electrophoresis
2100 Bioanalyzer
HiSens Assay
4.3 4.8 5.3 5.8 6.3 6.7 7.2 7.7 8.2 8.7
OFFGEL well pH
kDa
100
70
50
30
15
5
Isoelectric point (pI)
Mole
cu
lar
weig
ht
Protein clean up and labeling
Page 86
Análisis de variedades
de trigo
Wheat Type A Wheat Type B
Protein extraction
Alkylation with IAA
Acetone precipitated
Labeling at 10 ug/ul total protein (Bradford)
100 ug labeled protein fractionated pH3-10
Fractions undiluted analyzed with 250HSP-AssayIsoelectric point (pI)
Mole
cu
lar
weig
ht
Protein Biomarker Discovery and Validation
Cancer Cells
Blood Vessel Wall
High Abundant
Protein (Albumin)
Leached Protein
(Potential Biomarker)
Agilent RestrictedPage 87
Protein Biomarker Application
Fractionate Separate/LC Prep Identify
Reduction of complexity and enrichment
3100 OFFGEL
Agilent 1200
Series Liquid
Chromatography2100 Bioanalyzer
Agilent 6000
Series Mass
Spectrometry w/
HPLC-Chip
Quality
Control
Sample Prep Simplify/Deplete
Extraction of unique proteins from samples and simplify sample for analysis
Extract protein from
tissue or blood with
FFPE Protein Extraction
Solution or PPS Silent
Surfactant
Blood serum/plasma samples
(or CSF, urine) deplete high-
abundant proteins with
Multiple Affinity Removal
System
Fractionate,
concentrate, and
desalt proteins
with mRP-C18
Digest with
Proteomics
Grade Trypsin
Agilent RestrictedPage 88
OFFGEL Incremento de la sensibilidad en MSejemplo de trabajo
Incremento de 4x en la
detección de proteínas tras
el fraccionamiento con el
OFFGEL
OFFGEL
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 91
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 92
Agilent
Tecnologies ;
Stratagene
Agilent Technologies
Stratagene
QPCR y QRT-PCR Bioreativos
.
Page 94
Fundamentos del OFFGEL
Problemática
- Muestras complejas
- Rango dinámico de concentraciones muy amplio:
- Dificulta el fraccionamiento de proteínas de baja abundancia
Técnicas de Separación:
• Electroforesis convencional en gel 2D:
– Técnica tediosa
– Teñido del gel, recorte, extracción manual
– Poco reproducible, difícil de automatizar
• Electroforesis OffGEL:
– Técnica de prefraccionamiento nuevo patentado y desarrollado
conjuntamente por Agilent Technologies y Diagnoswiss
– Alta resolución y recuperación de analitos en disolución
Comparación de métodos para cuantificación
Page 95
Product training: QPCR Reagents Workflow
QPCR Reagents - Brilliant II
Sample Preparation
QRT-PCR Controls (QPCR from cDNA)
96
Absolutely RNA(Total RNA, FFPE & miRNA:high yields of highly pure total RNA)
SideStepIsolation Kits
AffinityScript RT and cDNA Synthesis Kits
QPCR Reference RNA(Human & Mouse)
Alien QRT-PCR Inhibitor Alert
Brilliant II SYBR and probe Master Mix• 1-Step and 2-Step QRT-PCR• FAST kits• High and Low ROX
Multiplex MM
miRNA Kit(target primers)
SideStep II QRT-PCR Kits
Page 97
• Miniprep, Microprep and Nanoprep Kits:
The Absolutely RNA Nanoprep Kit # 400753 is the only kit qualified to isolate
concentrated total RNA from the smallest samples, down to a single cell.
• 96 Microprep Kit:
High-throughput RNA purification kit in a 96 well format to cross sell with V1 (Bravo)
• FFPE Kit:
Non-toxic method to isolate quality RNA from paraffin embedded tissue sections
• SideStep isolation kits:
Quantitate gene expression directly from cell lysates
Samples ready for QRT-PCR in 10 minutes
KITS DE STRATAGENE QPCR Y QRT-PCR
Sidesteps
-Permite eliminar el paso previo de purificación (ADN, cADN,ARNm,ARN)
para la QPCR y QRT-PCR.
-Cuantificación de expresión génica en células sin aislamiento de RNA desde
(100 células a 1 millón de células).
-Lisis celular y estabilización de los ácidos nucleicos por encima de los 20
meses a (-80 ºC).
-Fácil de usar y en formato de un solo tubo lisis y estabilización de RNA
en 10 mins.
-No tóxico y evita precipatación no orgánica o etanol.
-Reactivos para el tratamiento con DNase.
-Primers testados (Quantos™ QPCR Normalization primers)
+Primer para DNA genomico humano
+ Normalizar número de células
+Amplificación de targets con número anormal de cromosomas.
KITS DE STRATAGENE QPCR Y QRT-PCR
Brilliant® -----------------------Reactivos tradicionales (SureStart).
Brilliant® II-------------------- Kits Alta sensibilidad { Taq polimerasa}.
Brilliant® Multiplex kit------Multiples reacciones de amplificación.
Brilliant® Sidestep kits----- Realización de QPCR y QRT-PCR a
partir de células lisadas.
Alien Inhibitor Alert kits----- Permite la detección de PCR inhibidores
en reacciones de QRT-PCR.
KITS DE STRATAGENE QPCR Y QRT-PCR
*Resultados consistentes y versatilidad.
*Compatibles con multiples tipos de deteccion química fluorescente:
-SYBR™ Green I dye
-TaqMan
-Molecular Beacons
-Others...
*Gran variedad de configuraciones de kits ….Optimizar la elección.
***Reference DYE (ROX).
-Normalización
-Control del pipeteo
-Variaciones entre pocillos(opcional en Mx)
-Comercializado en 2 formatos: -Separado (flexibilidad)
- HIGH and LOW rox
Page 101
SYBR® GreenSequence unspecific
dsDNA detection
Brilliant® II SYBR®
QPCR Mastermix
Brilliant® SYBR®
Core reagent kit
Regular Mastermixes
DNA quantification
2-step QRT-PCRRNA quantification
Includes AffinityScript™
QPCR cDNA synthesis kit
1-step QRT-PCR
RNA quantification
Brilliant® II SYBR®
QRT-PCR Mastermix
2-step
Brilliant® II SYBR®
QRT-PCR Mastermix
1-step
Brilliant® II
QPCR Mastermix
Probe based
detectionSequence specific
target detection
Brilliant® II
QRT-PCR Mastermix
2-step
Brilliant® II
QRT-PCR Mastermix
1-step
Brilliant®
Multiplex QPCR
Mastermix
Brilliant® II
Core reagent kit
1-step
Brilliant®
Core reagent kit
qPCR Reagent Evaluation – Qualifying Questions
102
1. Do you sometimes have low copy genes that you want to detect but can’t?
2. Do you have difficulty quantifying small differences in gene expression?
3. When performing quantification, are your results statistically significant? If
not, could be related to the reproducibility of replicates due to the master
mix performance.
4. Do you quantify template over a large dynamic range?
5. Do you work with small or limited amounts of RNA?
6. How often do you encounter genes with secondary structure that you
need to reverse transcribe and amplify? (a higher temperature RT may
help)
7. Would your research project benefit from shorter run times? (accomplish
more runs per day)
102
Brilliant III Ultra-Fast Benefits/Features
103
For faster, improved real-time quantitative PCR (qPCR) on StepOnePlusand CFX96 Real-Time PCR platforms – choose Agilent
Feature Benefit
New fast and highly processive
polymerase
Incorporates nucleotides during amplification faster
than standard Taq resulting in ~60% shorter run
times without compromising QPCR data quality
Novel hot start technology
Enhances the specificity by reducing the formation
of primer-dimers and secondary non-specific PCR
products
Targeted Master Mixes
New reagents validated across a range of fast real-
time PCR systems across a range of templates and
targets
Better Performance
Greater reproducibility and sensitivity within an
assay and across multiple assays and templates at
very low copy number
Brilliant III Ultra-Fast QPCR MM, – More Accurate
Quantification vs. Competitors
104
Dissociation curves comparing different QPCR SYBR
Green master mixes with primers specific for a 305bp
Numb-1 target, 50pg human gDNA template (in
quadruplicate). Brilliant III Ultra-Fast SYBR Green
Master Mix shows higher specificity with no primer-
dimer formation.
Company Q
Tm full prod: 83.1
Company A
Tm full prod: 78.6
Non-specific
amplification
products
Non-specific
amplification
products
Agilent Brilliant III
Tm full prod: 82.6
104
105
Company T generates earlier Cts, but the efficiency is compromised by formation of these artifacts which can compete with the
specific product.
Company Q
Company R
Company T
Agilent Brilliant III
Non-specific
amplification
products
Non-specific
amplification
products
Non-specific
amplification
products
Greater Degree of Confidence in Gene Expression data - Novel Hot Start
Technology of Brilliant III Ultra-Fast = absence of Non-Specific
Secondary Products
106
Brilliant III Ultra-Fast Master Mix generates Earlier Cts and Better
Reproducibility
GUS TBP CyclophilinRoche FastStart
Brilliant III Ultra-Fast
Eff.= 86% Eff.= 96% Eff.= 82%
Eff.= 99% Eff.= 93% Eff.= 98%
Brilliant III Ultra-Fast Master Mix generates Ct values ~8 cycles earlier than the competitor for the Cyclophilin target – this represents
>2 orders of magnitude difference in detection.
Improved Specificity over the leading competitor master mix
107
Brilliant III
Ultra-Fast
QPCR
Master Mix
ABI Fast
Universal PCR
Master Mix
Eff.= 94%
Eff.=79%
GUS primers/probe set (ABI Assays-on-Demand)
107
108
Improved Specificity over the leading competitor master mix
ABI Fast Universal Brilliant III Ultra-Fast
100 10 1 0.1 0.01 NTC 100 10 1 0.1 0.01 NTC
Primer-dimer
product
Input amount
108
109
Improved Specificity over the leading competitor master mix
Brilliant III
Ultra-Fast
QPCR
Master Mix
ABI Fast
Universal PCR
Master Mix
TBP primers/probe set (ABI Assays-on-Demand)
Eff.=85%
Eff.=100%
109
110
Improved Specificity over the leading competitor master mix
ABI Fast Universal Brilliant III Ultra-Fast
100 10 1 0.1 0.01 NTC 100 10 1 0.1 0.01 NTC
Primer-dimer
product
Input amount
110
miRNA Detection Juni, 2008
miRNA Detection
High Specificity miRNA QRT-PCR Detection Kit: Kit Performance
Discrimination between different let7 family members:
let-7 miRNA (1010 miRNA synthetic templates) were converted to cDNA:
Detection with each of the let-7 miRNA-specificprimers (miRNA-specific assay)
Copy number of the matching primer and templatewas 100%.
Copy number of a non-matching primer and templatedetermined as a percentage of the matching primerand template (% relative detection)
-Aprox 474/3000 miRNA humano
detecados.
-Importancia en detección de enfermedades
infecciosas, metabolomicas y cancer.
-Complejidad de estudio;
(longitud,homologia, el potencial de
degredación).
PCR Reagents Workflownew selection guide available !!
Site-Directed Mutagenesis
High-Fidelity PCR
Cloning
112
Pfu Ultra II HS(highest accuracy, Amplify up to 19 kb)
Herculase II Fusion(also for routine applications, Equivalent fidelity to Pfu)for 40 rxn)
StrataClone PCR Cloning kits(topoisomerase-based cloning,Mammalian Expression kits,All kits contain competent cells)
XL10-Gold Ultracompetent Cells, Provide 20-30 fold higher transformation efficiency of for large and ligated DNA
QuikChange Lightning &Lightning Muti-Site Kits,Generate mutants in less than 3 hours
Proteomic
MARS columns
Stratagene PCR Enzymes
Paq5000 and Paq5000 Hotstart DNA Polymerase
• Robust Taq substitute derived from a Pyrococcus species
• Easily substituted into standard PCR protocols
• Provided with a pre-optimized buffer
• Priced as low as 5 cents per unit (5,000 U; € 250.00)
• Equal to better yield with faster cycling conditions (30 sec/kb instead of typical 1
min/kb for standard Taq): shorter PCR run times
113
•Hot start version: economical
alternative to hot start Taq DNA
polymerases (5,000 U for €
750.00)
•Novel hot start technology for
increased specificity
•Convenient room temperature set
up
•Available in master mix format for
added convenience and
throughput: less pipeting steps
Stratagene PCR Enzymes
PfuUltra II HS DNA Polymerase
• Highest accuracy of any PCR
enzyme (only 1 error per 2.5 million
bases)
• High specificity hot start
formulation: ideal when amplifying
low copy number targets from
complex genomic DNA
• Reduced overall PCR extension
times: 70-80% quicker time-to-
results and increased throughput
• Extended length capability up to
19kb (long templates)
• Ideal for PCR cloning and site-
directed mutagenesis
114
Stratagene PCR Enzymes
L
Herculase II Fusion DNA Polymerase: high fidelity at low price
•Superior yields for routine PCR applications
•Amplify difficult templates, GC-rich, and from very low
starting amount
•Equivalent fidelity to Pfu DNA polymerase, ideal for PCR
cloning, RT-PCR and site-directed mutagenesis
AffinityScript RT-PCR Kit
One-Step
• Products useful for
cloning and sequencing
• Simple, rapid setup of
multiple reactions
• Minimizes contamination
in a single tube reaction
Two-Step
• Generates homogenous
population of cDNA
• Generate cDNA Libraries
• Recovery of specific
clones from a
heterogonous population
• First-strand cDNA for
downstream QPCR
specific for multiple genes
One Vs. Two-Step RT-PCR
AffinityScript One-Step RT-PCR Kit: #600188)
• Superior PCR product yields from up to 9.5 kb targets
• Cloning → Protein expression
• Sensitive detection from low copy genes
• Simple One-tube One-step reaction format from RNA to PCR
• Can be used for multiple reactions in the same tube simultaneously
• AffinityScript is the most versatile reverse transcriptase available with a
broad temperature range from 37° to 55°C promoting first strand cDNA
synthesis through GC rich RNA targets: better than SuperScript III based kits
• AffinityScript bundled with Herculase 2 Fusion DNA polymerase for
amplifying cDNA from challenging applications such as low RNA input and
low copy genes
Page 118
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Agilent Restricted
Month ##, 200XPage 119
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 120
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 121
Group/Presentation Title
Agilent Restricted
Month ##, 200XPage 122
MUCHAS
GRACIAS POR
SU ATENCIÓN
MUCHAS GRACIAS POR SU
ATENCIÓN