Realtime PCR just got better - agilent.com · Freedom of choice: Choose the filter configuration that suits your needs. Use all available QPCR ... Discrimination capabilities-500

Agilent’s

Mx3000P and Mx3005P™

Dr. Ivan Bendezu

Genomics Agent

Andalucia

Realtime PCR just got better

June, 2008Page 2

Intro to QPCR and Instrumentation

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SYBR Green:

Dye attaches to the minor groove of

double-stranded DNA

Very low fluorescence when not bound

1000x increase in fluorescence when

bound to DNA

Sequence unspecific:

All double-stranded molecules are

detected (specific amplicon, primer

dimers etc.)

SYBR green is a good beginners chemistry and

most valuable in assay validation and for assay troubleshooting

Real-time PCRChemistries – SYBR Green

June, 2008Page 3


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unbound probefree in solution

Light emission

Light

energy transfer

Taq

Reporterdye

Acceptordye (Quencher)

Taq

Light emissionLight

Taqman Chemistry:

Uses exonuclease activity of Taq

in combination with a dual-labeled

oligonucleotide – the probe.

Higher specificity: Detection only

if primer and probe are bound on

the same strand

Allows multiplexing of multiple

target sequences in the same

reaction

Real-time PCRChemistries – Taqman

June, 2008Page 4


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Thermal system

Hot top

Tungsten/Halogen Lamp

Emission filter wheel

Excitation filter wheel

Fiber optic

cable and

scanning arm

Real-time PCRInstrumentation

Mx3000P/Mx3005P

June, 2008Page 5


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Built for multiplexing:

Superior performance in multiplexing QPCR applications

Flexible measurement options:

ROX channel available for target detection as no reference

dye is needed to correct for positional effects.

Mx3005P is able to mismatch excitation and emission

filters.

Excellent block uniformity:

Highly uniform results across the block allow robust and

low variance measurements for reliable and significant results

5-plex data

50 copies of

target

Dyes: Atto425, FAM,

HEX, TxRd, Cy5

Protein melt curve using

SYPRO Orange (ex: FAM

em: ROX)

June, 2008Page 6


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Over 10 years of experience in real-time

QPCR instrumentation

Freedom of choice: Choose the filter configuration

that suits your needs. Use all available QPCR

chemistries.

Halogen lamp based excitation

Scanning optical design: Avoids positional effects

4 colors (Mx3000P™) or 5 colors (Mx3005P™)

Photomultiplier tube allows sensitive measurement

Internal computer saves data: Recovery of data

in case of external computer failure


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June, 2008Page 7

Real-time PCRInstrumentation – Thermal system

• Temperature uniformity:

(+/- 0.25°C @ 72°C across the block).

• Solid state, Peltier-based thermal

system

• Ramp rate: up to 2.5°C/s

• Hot Top (Heated lid).

Mx3005

others

Variation of amplicon TmHerrmann et al., Clin. Chem. 52 (2006) and 53 (2007)


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June, 2008Page 8

Real-time PCRInstrumentation - Optics

Halogen lampExcitation range

350-750 nm

Excitation filter wheel4 or 5 filters with narrow

bandpass (~10 nm)

Fibre optics cable

Emission filter wheel4 or 5 filters with narrow

bandpass (~10 nm)

PhotomultiplierHighly sensitive

detection of

fluorescence

Simultaneous vs scanning

illumination and detection

Asynchronous (scanning)

Constant intensity(No positional effects)

Simultaneous (CCD)

Intensity depends on well position

Lamp

Higher intensity

lower intensity

3.5 s scanning time per dye

June, 2008Page 10


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Alx350 FAM

TET HEX

JOE ROX Cy5Cy3

TAM TxRd

Excitation range: 350 to 750 nm

Emission range: 350 to 700 nm

Standard filter set on Mx3000P®Standard filter set on Mx3005P®Available filters can be used with alternative dyes

Freedom of choice: Choose the filter combinations that match your needs!

Atto425

Real-time PCRInstrumentation - Detected Dyes

June, 2008Page 11


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Optimized Filter Sets

Designed for multiplexing - Discrimination capabilities

-500

0

500

1000

1500

20002500

3000

RFU

FAM HEX ROX Cy5

FAMHEXROXCy5

350 nm 750 nm

Excitation Filter10 nm Interference

Em. Filter

Real-time PCRInstrumentation - Optics

Adaptive Baseline Algorithm

Adaptive baseline correction

Adaptive Baseline Corrects for Drift

RdR

X

Moving Average Algorithm

• Random noise in

baseline is removed to

make it easier for sw to

find exponential amp

• smoothed by replacing

each data point with

the average of the

points around it. (user

selectable, 3 pt default)

• Minor impact on Ct

Optional Data Smoothing

3 Pt Smoothing

MxPro Software:Flexible and easy to use

MxPro Experiment

Types

Melt curve analysis

Gene expression

Probe based Quantification

Real-time AD/SNP analysis

Determine opt annealing temp

RNA and DNA quantitation

End-point Allele Discrimination

Experiment types can be converted

after data collection

Now featuring Multiple experiment

Analysis: Up to 12 plates!

Software OrganizationFollows Experimental Workflow

“Run Status” Box shows

run time parameters

QPCR Assay QC & RNA Quality to Expression Levels

(Application Note RNA QC/QPCR (5989-7730EN)

oligo-dT

random

GAPDH 3‘ assay: Expected size 126 bp

121 bp

20

RIN 8.9

0 minA RIN 6.5

30 minB

RIN 4.6

45 minC RIN 2.3

75 minD

-14

-12

-10

-8

-6

-4

-2

0

RIN 8.9 RIN 6.5 RIN 4.6 RIN 2.3

GAPDH 5' assay

GAPDH 3' assay

G

A

P

D

H

DNA Chip to check QPCR amplicon product size RIN # corresponding expression level changes in QRT-PCR

June, 2008Page 21


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Real-time PCRApplications

Sensitive sequence

detection and

quantification

Gene expression

analysis

Detection of

sequence variants

Fluorescence

detection

Pathogen detection

GMO analysis

Quality control

Forensics

Validation ofmicroarrays

Comparativequantification

ChIP

Copy number analysis

CGH

miRNA detection

SNP detection/

allelic discrimination

Methylation studies

Immuno-PCR:

Detect proteins with

QPCR

DNA/RNA quantification

Protein stability testing

Reporter gene activity

Versatile technology:

and many more …..


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June, 2008Page 22

QPCR related reagent product portfolio

RNA extraction

Reverse Transcription

Controls

QPCR

Absolutely RNA®:

RNA extraction from

cells and tissue

SideStep™:

Cells to PCR

AffinityScript™:

Multi temperature

Reverse transcriptase

Brilliant® II MM:

For SYBR green and

probe.

High Specificity

miRNA Detection kit:

miRNA detection by

QPCR

Alien® Inhibitor Alert:

Inhibition control and

external spike-in for

normalization

QPCR reference

RNA:

External reference

sample for human and

mouse

June, 2008Page 23


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QPCR assay validation

QPCR(microarray validation,

downstream studies)+ Brilliant® reagents

Ways of Working:Combining the strengths of 3 platforms

Microarrays(expression, CGH, copy number)

Nucleic acid quantification

and QC

Sample PreparationAbsolutely RNA®

SideStep™

June, 2008Page 24


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Thanks for your attention!

Documents

Realtime PCR just got better - agilent.com · Freedom of choice: Choose the filter configuration that suits your needs. Use all available QPCR ... Discrimination capabilities-500