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
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
Agilent Restricted
Real-time PCRInstrumentation
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
Intro to QPCR and Instrumentation
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Real-time PCRInstrumentation
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
Intro to QPCR and Instrumentation
Agilent Restricted
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)
Intro to QPCR and Instrumentation
Agilent Restricted
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
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
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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 …..
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
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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
Intro to QPCR and Instrumentation
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Thanks for your attention!