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Complete Solutions for Real Time PCR
Complete Solutions for Real Time PCR
Complete Solutions for Real Time PCR
• MyiQTM and iCycler iQTM Specifications
• PCR Techniques, Application examples
• Reagents and Kits
Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyDr. Luis Ugozzoli, Bio-Rad Laboratories Hercules, CA
Complete Solutions for Real Time PCR
iCycler iQ® and MyiQTM
- are based on the iCyclerTM base unit with a large high resolution on-board VGA display and intuitive software
- software updates are offered by CD or internet
- 96 well reaction module for real-time PCR(various reaction modules for iCycler conventional PCR)
- 96 well reaction module with thermal gradientfor optimization of PCR in real-time PCR
- iCycler upgrade for-real time PCR with theoptical module iQ or MyiQ
iCycler
Complete Solutions for Real Time PCR
384-well
2x48-well
96-well Reaction Module
60-well
Thermal - Gradient ReactionModule
96-well Reaction Module
Thermal - Gradient ReactionModule
66.0° C65.2° C63.9° C61.8° C59.0° C57.1° C55.8° C55.0° C
back
front
[ Mg ++ ] [ Primer ] [Enzymes]
Thermal - Gradient ReactionModule
Dynamic Ramping enables that the different temperatures within the thermal gradient are reached at the same timepoint.
72 °C
95 °C
66 °C
61.8 °C
Filter Fluorescence DetectionWavelenghth specific exitation of different fluorophores and theircorresponding emission light is measuerd by a filter basedtechnology.For each fluorophore, a paired excitation and emission filter isused
The optical module iQfor real-time-PCR
CCD
Tungsten-Halogenlight source
Excitation filters96 well plate
IntensifierEmission filtersEmission light mirror
Exitation-light mirror
The MyiQ single color real-time-PCR system
MyiQ generates the same great data quality as the iQ system
Two-fold dilution series of genomic DNA
MyiQ generates the same great data quality as the iQ system
Uniformity Experiments:50 µL FAM uniformity CT = 21.41 ± 0.0950 µL SYBR uniformity CT = 19.17 ± 0.1215 µL SYBR uniformity CT = 18.71 ± 0.13
Fluorescence Detection
Sample
Sample
20 21 22
Cycle
Cycle
Sample
Fluorescence Probes
• Cleavage Based Assay - TaqMan® Assays• Displaceable Probe Assays
– molecular beacons– Dual oligo FRET probes
• Probes incorporated directly into the primers– Amplifluor™– Scorpions™
Complete Solutions for Real Time PCR
• MyiQTM and iCycler iQTM Specifications
• PCR Techniques, Application examples
• Reagents and Kits
Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyPh.D. Luis Uggozzoli, Bio-Rad Laboratories CA, Herkules
4 Target Multiplex Multicolor - PCR
FAM/Tubulin
Tex Red/β-Actin
HEX/Cyclophilin
Cy5/GAPDH
Cycle
log
rela
tive
Flu
ores
cen
ce
Texas RedFAM
Hex
Cy5
10
100
1000
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49
Tubulin
Cyclophilin
beta Actin
GAPDH
Konzentration (10n)
Thre
shol
d C
ycle
Tubulin r2 = 1.000y = -3.44x + 36.82
GAPDH r2 = 0.996y = -3.34x + 37.29
β-Actin r2 = 0.998y = -3.55x + 41.07
Cyclophilin r2 = 1.000y = -3.40x + 37.04
4 Target Multiplex Multicolor - PCR
SNP Detection with FRET ProbesWild type Mutant
Heterozygote
iCycler iQ Software(Data Analysis)
PCR Quantification Demonstration ReportPCR Base Line Subtracted Data
Contains All Available Data
Current Date : Monday, June 25, 2001CurrentUser: User1Run Date: Wednesday, May 23, 2001Run User: User1
OPD File Name: beta_act1.opdOPD File Path: C:\Programme\Bio-Rad\iCycler\User1Use calibration (RME) data from OPD? Yes
Plate Setup File Name: beta_act2.ptsPlate Setup File Path: C:\Programme\Bio-Rad\iCycler\User1Protocol File Name: test.tmoProtocol File Path: C:\Programme\Bio-Rad\iCycler
Comments:beta Actin mit TaqMan Probe und mit SYBR I.Anschließende Schmelzkurvenanalyse auf Primerdimere.
Filter Wheel Setup: FilterSet4
Protocol::Protocol[]:
Standard Quantity Units: Copy Number
PCR Quantification Demonstration ReportPCR Base Line Subtracted Data
Contains All Available Data
Selected Fluorophore: FAM-490
Run Parameters
Hot Start? NoTemperature Control Mode: Algorithm Volume: 50.00 ulWell Factor Collection Mode: Experimental Plate
Data Analysis Parameters
Display ControlsPCR Display Mode (Single Point/ All Candidates): Single Point
Data SelectionPCR Reading Selection (Analysis/Collect): Analysis EnableData Window Size: 10.00%Center: end
PCR Amp/Cycle Chart
Complete Solutions for Real Time PCR
• MyiQTM and iCycler iQTM Specifications
• PCR Techniques, Application examples
• Reagents and Kits
Dr. Marcus Neusser, Bio-Rad Laboratories, GermanyPh.D. Luis Uggozzoli, Bio-Rad Laboratories CA, Herkules
Complete Solutions for Real Time PCR
Products for PCR and real-time PCR– iTaq™ DNA Polymerase– dNTPs– iQ™ Supermix, iQ™ SYBR® Green Supermix
iTaq™ SYBR Green Supermix with ROXProducts for Reverse Transcription
– iScript™ cDNA Synthesis Kit– iScript™ One-Step RT-PCR Kit with SYBR Green– iScript™ One-Step RT-PCR Kit for probes
iScript is UNIQUE
Bio-Rad’siScript cDNA Synthesis Kit
5' 3'mRNA 5'3'
First strand cDNA synthesis
Second strand cDNA synthesis
cDNA amplification
cDNA3' 5'
3'5'
5'3'
3'5'
5'3'
3'5'
5'3'
3'5'
5'3'
• RNA template• DNA primers
- gene-specific primers- oligo d(T) primers- random primers
• First Strand cDNA synthesis using Reverse Transcriptase (AMV, MMLV, Tth, etc.)
• Second strand cDNA synthesis and cDNA amplification using standard PCR conditions and enzymes
One-Step vs. Two-Step RT-PCROne-Step RT-PCR Two-Step RT-PCR
Primers + RNA
Add all reagents For RT-PCR:• reverse transcriptase• DNA polymerase• dNTPs• universal buffer
• RT (50oC, 1hr)• PCR
Primers + RNA• 70oC for 2min• Cool on ice
Add RT reagents
• RT (42oC, 1hr)
Dilute sample into new tube
Add PCR reagents
• PCR
iScript is EASY TO USE
iScript cDNA Synthesis KitTo a 0.2-ml tube add:
4 µl 5X cDNA Synthesis System1 µl iScript enzyme mixx µl nuclease-free waterx µl RNA sample20 µl final volume
OTHERTo a 0.2-ml tube add:
0.5 µl oligo(dT)12-18, 500 mg/ml0.5 µl Random hexamers, 50 ng/ml2 µl 10X buffer4 µl 25 mM MgCl21 µl 10 mM dNTP2 µl 0.1M DTT1 µl RNaseOUT (40U/µl)1 µl rt enzyme (50 U/ml)x µl DEPC-treated waterx µl RNA sample20 µl final volume
iScript is significantly FASTERiScript cDNA Synthesis Kit
5 min at 25oC 30 min at 42oC5 min at 85oCHold at 4oC
Total time: 45 minutes
With iScript – just set-up the thermal cycler and walk away!
OTHER
5 min at 65oCQuick chill on iceAdd more itemsMix and incubate for 2 min at 42oC Add RT enzymeIncubate for 50 min at 42oCHeat kill 15 min at 70oC
Total time: 85 to 90 minutes
With OTHER, you’re tied to the bench AND you’ll be waiting for your cDNAlonger!
PRODUCT PERFORMANCE DATA
Sensitivity of the iScript cDNASynthesis Kit with iTaq DNA
Polymerase for RT-PCR
M 10fg 100fg 1pg 10pg 100pg 1ng 10ng control
Reliable results across a broad range of input concentrations
iScript qRT-PCR Standard Curve Comparison: cDNA serial dilution vs. total RNA serial dilution
Log Starting Quantity (femtograms of input RNA)
Note: 1/10th of cDNA reaction used for PCR
1 2 3 4 5 6 7 8 9
ye
d u
T
5
10
15
20
25
30
35
40 cDNA Standard Total RNA Standard
cDNA total Slope -3.394 -Corr. Coef. 0.999 0.999 Intercept 38.91 3809
Complete and unbiased coverage of the RNA input population
Reagents and KitsPerformance Testing of iTaq
With Antibody Without Antibody
iScript can be used with all of Bio-Rad’s DNA polymerase products
B. iQ Supermix
C. iQ SYBR Green Supermix
A. iTaq DNA PolymeraseM 10fg 100fg 1pg 10pg 100pg 1ng 10ng control
1µg100ng10ng1ng100pg10pg1pg
Reagents and Kits iQ Supermix
Catalog # Description
170-8851 iQ ™SYBR Green Supermix w. ROX, 500 x 50 µl reactions170-8852 iQ ™SYBR Green Supermix w. ROX, 1000 x 50 µl reactions
170-8850 iQ™ SYBR Green Supermix w. ROX, 200 x 50 µl reactions170-8864 iQ ™Supermix, 1000 x 50 µl reactions170-8862 iQ ™Supermix, 500 x 50 µl reactions170-8860 iQ™ Supermix, 100 x 50 µl reactions
170-8880 iQ™ SYBR Green Supermix, 100 x 50 µl reactions170-8882 iQ ™SYBR Green Supermix, 500 x 50 µl reactions170-8884 iQ ™SYBR Green Supermix, 1000 x 50 µl reactions170-8890 iScript™ c-DNA Synthesis Kit, 25 x 20 µl reactions170-8884 iScript ™c-DNA Synthesis Kit, 100 x 20 µl reactions
www.bio-rad.com/amplification
