2100 Bioanalyzer Assays and Applications

Page 18

RNA Applications

RNA QA/QC Microarrays

Gene Expression

RNA QA/QC qPCR

RNA QA/QC mPCR

smallRNA QA/QC

Page 19

2008

RNA QC WorkflowAnalysis of Total RNA Integrity

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2008

RIN 9.2

RIN 5.8

RIN Application – Assessment of RNA Integrity

Intact RNA: RIN 10

Partially degraded RNA: RIN 5

Flu

ore

scence

5

6

7

8

9

18

S

28

S

Flu

ore

scence

0

5

10

15

20

25

30

35

40

45

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2008

Partially degraded RNA: RIN 5

Strongly Degraded RNA: RIN 3

18

S

28

S

Flu

ore

scence

0

1

2

3

4

5

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

RNA extraction

Workflow: QPCR with Bioanalyzer QC

RNA degradationRNA 6000 Nano kit

Stratagene Absolutely RNA®

MiniPrep kit

RNA QC and quantification

Reverse transcription

QPCR assay validationQPCR

3/9

DNA 1000 kit

Stratagene AffinityScript™ Agilent 2100

bioanalyzer

RNA QC/qPCR Sales Tools

•RNA integrity/qPCR CD 5989-9911EN - Distributed more than 3000 CDs worldwide

• Publications, References

• Application Notes

• Product Information

• Weblinks• Weblinks

• RNA QC/qPCR Application Note (5989-7730EN)

• Customer Presentation

• External References and publications

External References – qPCR News (e-Newsletter)

qPCR e-Newsletter May Issue:

Focus on RNA quality control

Links to list of RNA integrity publications

MIQE: Minimum Information for publication Information for publication of qPCR experiments

Almost all listed publications

refer to the Bioanalyzer for

RNA QC

A

B

Laser Microdissection – PALM MicroBeam System

and RNA Pico kit

Laser

microdissection

Laser pressure

200 pg 200 pg

total RNA

Page 25

2008

Laser Microdissection and Pressure Catapulting

(LMPC)

RNA extraction

B

C

RNA sample QC using the Agilent 2100 bioanalyzer and

the RNA 6000 Pico LabChip kit

Data kindly provided by P.A.L.M. Microlaser Technologies

Laser pressure

catapulting:

Section after

catapulting of

selected area

Catapulted area

in the collection

device

18S

28S

24 29 34 39 44 49 54 59 64 69

Sm

all

RN

A R

eg

ion

New Small RNA Assay versus existing RNA Assay

18s

28s

RIN: 8.1

Lo

wer

Mark

er

RNA 6000Nano kit

Small RNA Kit

RNA 6000Nano

Size range: 25-6000nt

Results: Integrity, Total RNA amount, gDNA contamination

Page 26

2008

miR

NA

Re

gio

n

Lo

wer

Mark

er

Small RNA Region

tRN

A

Small RNA Kit

NEW! Small RNASize range: 6-150nt

Results: miRNA amount, Ratio and amount of other Small RNA

5s

5.8

s

DNA Applications

mPCRanalysis

Restriction Digest

Gene Expression

Food Analysis

ForensicTesting

Page 27

2008

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

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

Determination of PCR Product Impurity

1*

2 3 4*

Flu

ore

sce

nce

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%

300 bp

Page 28

2008

1*

2 3 4 5 6 7*

Flu

ore

sce

nce

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 : > 50%

3000 bp

Time (seconds)

Quantitative data from Agilent 2100 bioanalyzer

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

Optimization of Multiplex PCR on a 19-plex PCR

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2008

Data kindly provided by QIAGEN GmbH, Germany

DNA QC in Next-Gen Sequencing - Situation

�Next Generation Sequencing is an emerging technology.

�Quality of sequences is one of the chief technical concerns about next gen

sequencing platforms in a recent customer survey by Insight pharma reports

�Leading next generation sequencing vendors (Illumina, Roche, Life

Technologies) recommend existing Bioanalyzer DNA assays in their workflows

� Bioanalyzer DNA kit: ~30% growth in FY08

�Growth potential: still NGS users who just use slab gel and Nanodrop�Growth potential: still NGS users who just use slab gel and Nanodrop

High Sensitivity DNA Kit

May 2009

Next Generation Sequencing WorkflowsBioanalyzer Quality Control officially recommended by: Roche - Illumina - ABI

Recommendation: use of RNA and DNA solutions by Agilent 2100 Bioanalyzer

Key Scientific Reference:

DNA QC in Next-Gen Sequencing – Illumina DNA

Library Prep Workflow

Simplified Illumina GAII Workflow

High Sensitivity DNA Kit

May 2009

New High Sensitivity DNA Kit - Product Structure

�Bioanalyzer kit for sizing and quantification of dsDNA fragments and dsDNAsmears in the 50 – 7000 bp size range down to pg/µl sensitivity

�Product structure follows standard DNA kit structures

�10 Chip Kits (11 samples/chip)

� SW upgrade to 2100 expert B.02.07 required

Agilent High Sensitivity DNA Kit

(5067-4626)

High Sensitivity DNA Reagents

(5067-4627)

High Sensitivity DNA Kit

May 2009

Protein Applications

Protein Purification

Protein Production

ProteinExpression

Food Analysis

Purity and QA/QC

Page 34

2008

Quality Control of Antibodies

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

Page 35

2008

90 kDa 160 kDa

Absolute Quantitation of IgG samples

Bioanalyzer: Protein Kit portfolio

Protein 80 kit

Prod No 5067-1515

Sizing: 5 – 80 kDaProtein staining on ChipSensitivity: Coomassie (ng/µL)25 Chip Kit (Max 250 samples)

Access to Small ProteinsQuick sample prepEasy to use Antibodies

Protein QA/QCAbundant Proteins

Standard Kits (P80,P230) for fastest analysis in average concenration range

High Sensitivity Kit (HSP-250) for pg amounts and extra wide linear quantification ranges

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2008

Protein 230 kit

Prod No 5067-1517Sizing: 14 – 230 kDaProtein staining on ChipSensitivity: Coomassie (ng/µL)25 Chip Kit (Max 250 samples)

Standard protein rangeQuick sample prepEasy to use

HSProtein 250 kit

Prod No 5067-1575Sizing: 10 – 250 kDaCovalent labeled ProteinsSensitivity: Silver (pg/µL)10 Chip Kit (Max 100 samples)

Highest Sensitivity4 Orders linear rangeLabeling reaction included

Abundant ProteinsExpression OptimizationRecombinant Proteins

Low abundant ProteinsImpurity analysisAntibodiesProtein QA/QC

Cell Applications

Protein Expression

Transfection Monitoring

Apoptosis Detection

Gene Silencing

Cell StainingInside / outside

Page 37

2008

Video