overview agilent 2100 bioanalyzer solutions lab on a ... · celular y proteínas (western)...

Overview Agilent

Lab on a

ChipTechnology

2100 Bioanalyzer Solutions

Cordoba 8 de Abril

Agilent 15 Marzo 2010

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.


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:


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


The LabChip® Approach - Simplified Model

＋

_+

-


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


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


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

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



Gel view and electropherogram view

→time/size

↑ fluorescence intensity/

quantity


0. preparatory transformations

• Noise reduction

(Savitzky-Golay filtering

• Baseline correction

• Spike rejection

Baseline correction disabledBaseline correction enabled


1. Peak recognition

table of

fluorescence

intensities

table of

peaks

Result table in Expert

Electropherogram in Expert


2. Alignment to markers

Lower marker

Upper marker

Analysis turned off –

Unaligned data

Analysis turned on –

Aligned data


3. Sizing

size:

ladder


4. Quantification (DNA/Protein samples)

peak area

concentration

upper marker peak area

(known concentration)

assay-specific calibration curve

(standard areas, see assay properties)

Ladder


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

Agilent Web pages with 2100 content

www.opengenomics.com

www.agilent.com/chem/labonachip

tAgilent 15 Marzo 2010

Aplicaciones con tecnología

microfluidica en ARN, ADN, Contaje

Celular y Proteínas (Western)

Group/Presentation Title

Agilent Restricted

Month ##, 200X

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.

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


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


6000 Nano 6000 Pico Small RNA

HSP 230

High Sensitivity DNA Cell Check Out



Agilent Restricted

Month ##, 200X

RNA Applications

RNA QA/QC

for

Microarrays

Gene

Expression

Genomic

DNA

ContaminationRNA QA/QC

for qPCR

RNA QA/QC

for mPCR

smallRNA

QA/QC


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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.)


Agilent Restricted

Month ##, 200X

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!!


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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.


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

DNA Applications

mtDNA

Screening

qPCR

validation,

impurity check

mPCR

validation,

impurity check

Restriction

Digest

Analysis

Gene

Expression

Oncology

Food

Analysis

Clinical

Research

Forensic

Testing


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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

PCR Product Analysis using the

DNA 7500 or DNA 1000 Kit

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

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

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

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

Restriction Fragment Analysis

Mutation Detection by RFLP

Wild type Mutant

Point mutationRestriction fragment cleavage site

Restriction

enzyme

digestion

+

2 fragments 1 fragment

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

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

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

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)


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

Cell Applications

Protein

Expression

Monitoring

Intracellular

Transfection

Efficiency

Monitoring

Apoptosis

Detection

Gene

Silencing

Protein

Expression

Monitoring

Cell Surface

Antibody

Staining


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

:

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


Agilent Restricted

Month ##, 200X

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





Agilent Restricted

Month ##, 200X

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


Agilent Restricted

Month ##, 200X

GFP On-Chip Staining - Histogram Quality

2100 bioanalyzer

Flow

Cytometer

¿Qué es la Proteómica?

-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!!

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

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

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

OFFGEL Bioanalyzer

¿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?

- 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)

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


Agilent Restricted

Month ##, 200X

Protein Applications

Antibody

QA/QC

Protein

Expression

Protein

Purification

Food

Analysis

Protein

QA/QC

Compliant

Protein &

Antibody

QA/QC


Agilent Restricted

Month ##, 200X

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

Expresión de proteínas

Protein 230 kit

Purificación de proteínas

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

Análisis de la capacidad de las columnas

Protein 230 kit

Análisis de anticuerpos

High sensitive

Protein 250 kit

Glicosilación de proteínas

Protein 230 kit

Control de calidad de Anticuerpos

Protein 230 kit


Agilent Restricted

Month ##, 200X

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

Protein 80 kit

Immunoprecipitation/Western Blot:

IP/HSP-250

Bioanalyzer

High sensitive

Protein 250 kit

Geles en 2D: Combinación de Offgel y

Bioanalizador

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

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 Restricted

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 Restricted

OFFGEL Incremento de la sensibilidad en MSejemplo de trabajo

Incremento de 4x en la

detección de proteínas tras

el fraccionamiento con el

OFFGEL

OFFGEL

MUCHAS

GRACIAS POR

SU ATENCIÓN

MUCHAS GRACIAS POR SU

ATENCIÓN

[email protected]


Agilent Restricted

Month ##, 200X

Agilent

Tecnologies ;

Stratagene

Agilent Technologies

Stratagene

QPCR y QRT-PCR Bioreativos

.

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

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

• 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.


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.


*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

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


QRT-PCR Mastermix

2-step


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


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


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


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


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


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


Agilent Restricted

Month ##, 200X


Agilent Restricted

Month ##, 200X

MUCHAS

GRACIAS POR

SU ATENCIÓN

MUCHAS GRACIAS POR SU

ATENCIÓN

[email protected]

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