Download Next Generation Quantitative PCR

Next Generation Quantitative PCR
Real-Time PCR or quantitative PCR (qPCR) is a PCR-based technique that is able to simultaneously amplify and detect changes in the amplicon
concentration. Real-Time PCR collects data during PCR amplification by utilizing fluorescence signal emitted by either DNA binding dyes such
as SYBR Green Dye, or special probes such as TaqMan probe. abm selection of EvaGreen qPCR MasterMixes and TaqProbe qPCR MasterMixes provides various options for both methods which also accommodates most qPCR thermal instrument models available on the market.
EvaGreen qPCR MasterMixes
abm’s EvaGreen 2X qPCR MasterMix provides all ingredients
Reverse Transcriptase (RTase) is a well-known potent inhibitor of
necessary for quantitative PCR in a premixed and optimized format.
PCR. abm’s EvaGreen 2X qPCR MasterMix outperforms competi-
A new generation of SYBR Green I, the EvaGreen Dye used in our
tors with its greater range of compatibility to commercially available
EvaGreen qPCR MasterMixes works in a similar mechanism
RTases. Compared to other leading brands, our OneScript® and
(Figure1). Available with either ROX or fluorescein as the internal
OneScript® Plus RTases also demonstrate the least amount of inhibi-
passive reference dye, abm’s EvaGreen 2X qPCR MasterMix offers
tion to downstream qPCR applications (Figure 2A-E).
unparalleled performance in sensitivity, signal-to-noise ratio, and
complete elimination of primer dimers.
• Streamlined protocol in a simple reaction set-up
• Accurate quantification of a variety of gene targets
abm's OneScript®
abm's OneScript® Plus
LifeTech's SuperScript III
NEB's M-MuLV H Minus
Thermo's Maxima
• Reduced risk of contamination with less pipetting steps
• Compatibility with most real-time PCR instruments
Denaturation
Polymerization
Forward primer
Figure 2A: With abm’s EvaGreen 2X qPCR MasterMix
abm's OneScript®
abm's OneScript® Plus
LifeTech's SuperScript III
NEB's M-MuLV H Minus
Thermo's Maxima
Reverse primer
Polymerization
Completed
Figure 2B: With ABI Power SYBR Green Master Mix
Figure 1: EvaGreen method: When the DNA polymerase synthesizes the
complementary strand to the template, the intercalating dye, EvaGreen,
incorporates itself into the dsDNA and emits a strong fluorescent signal which
can be detected by the thermal cycler.
abm's OneScript®
abm's OneScript® Plus
LifeTech's SuperScript III
NEB's M-MuLV H Minus
Thermo's Maxima
Figure 2C: With Biorad iQ SYBR Green Supermix
Next Generation Quantitative PCR
abm's OneScript®
abm's OneScript® Plus
LifeTech's SuperScript III
NEB's M-MuLV H Minus
Thermo's Maxima
Figure 2D: With Takara SYPR Premix Ex Taq II
abm's OneScript®
abm's OneScript® Plus
LifeTech's SuperScript III
NEB's M-MuLV H Minus
Thermo's Maxima
Figure 2E: With Roche FastStart SYBR Green Master
Figure 2 (A-E): 20 μl RT reactions (100 pg of Human RNA per reaction) were performed with various RTases available on the market. 1 μl of each RT product
was then used directly as template for GAPDH amplification using qPCR MasterMixes from various leading brands.
abm’s EvaGreen 2X qPCR MasterMix is the ultimate solution for reducing non-specification amplification in qPCR. Its optimized buffer system
works in tandem with a highly efficient HotStart DNA polymerase to minimize signal-to-noise ratio (Figure 3A-E)
Figure 3A: abm’s EvaGreen
Figure 3B: ABI Power SYBR
Figure 3C: Biorad iQ
Figure 3D: Takara SYPR
2X qPCR MasterMix
Green MasterMix
SYBR Green SuperMix
Premix Ex Taq II
Figure 3 (A-E): Melt curves of the corresponding MasterMixes from the reactions shown in Figure 3A-3E.
Figure 3E: Roche FastStart
SYBR Green Master
EvaGreen Express qPCR MasterMixes
abm’s EvaGreen Express 2X qPCR MasterMix provides all ingrediActivation Amp. cycle
ents necessary for extremely rapid quantitative PCR in a premixed
and optimized format. Our proprietary TaqFast DNA Polymerase
shortens reaction time to 1/5 of standard qPCR time. Available with
EvaGreen Express
2X qPCR
either ROX or fluorescein as the internal passive reference dye,
95°C
30 s
Melt. Curve
95°C
1-5 s
1X
40X
60°C
10 s
1X
abm’s EvaGreen Express 2X qPCR MasterMix offers unparalleled
sensitivity, signal-to-noise ratio, and complete elimination of primer
Activation
dimers.
• Extremely rapid activation – only 30 seconds!
• Ultra-fast qPCR thermal cycling – 10 second annealing/elongation time
• Streamlined protocol with a simple reaction set-up
• Ultra-fast quantification of a variety of gene targets
• Reduces pipetting steps to minimize the risk of contamination
• Compatible with most real-time PCR instruments.
EvaGreen
2X qPCR
Standard protocol
95°C
10 m
Amp. cycle
Melt. Curve
95°C
15 s
60°C
60 s
1X
40X
1X
Next Generation Quantitative PCR
TaqProbe qPCR MasterMixes
One-Step EvaGreen/TaqProbe qRT-PCR Kit
abm’s TaqProbe 2X qPCR MasterMix is designed for high through-
Convenient real-time RNA quantification in one step.
put quantitative PCR using TaqMan
®
probe-based chemistry.
(Figure 4) Available with either ROX or fluorescein as the internal
passive reference dye, abm’s TaqProbe 2X qPCR MasterMix offers
superb performance in sensitivity and signal-to-noise ratio. The
multiplex formulation supports quantitative amplification and
detection of up to four targets simultaneously with consistent and
reliable results.
• Improved fidelity and yield for reverse transcription
• Prevention of template (RNA) degradation with RNaseOFF
Ribonuclease Inhibitor
• Superb performance with respect to sensitivity and signal-to-noise
ratio
• Significant reduction in non-specific PCR amplification by
utilizing HotStart Taq DNA polymerase
• Streamlined protocols with its single tube set-up
• Fully optimized for detection of low-copy genes
• Accurate quantification of a variety of gene targets
- high-quality, full-length cDNA from as little as 0.01pg of RNA
• Reduce contamination risks with less pipetting steps
• Streamlined protocol in a simple single-tube reaction set-up
• Compatible with most real-time PCR instruments
• Reduced risk of contamination with less pipetting steps
Forward primer
R
Probe
Q
One-Step qRT-PCR Kit
Polymerization
Reverse primer
R
Q
+ RNA
+ Gene-Specific Primer
+ qRT-PCR Enzyme Mix
+ EvaGreen / TaqProbe
qPCR Mastermix
1
qRT-PCR
abm G471-X
G493-X
One-Step
qRT-PCR
Strand displacement
R
Q
Cleavage
R
Q
Polymerization
Completed
Figure 4: TaqProbe method: 5’ to 3’ exonuclease activity of Taq DNA polymerase
displaces the reporter dye of the TaqMan Probe, and cleaves the reporter at the
5’ end from the quencher at the 3’ end to emit fluorescence
Figure 5 : 20 μl One-Step qRT-PCR reactions (20 ng, 2 ng, 200 pg, 20 pg or 2 pg of RNA
per reaction) were carried out with abm’s One-Step EvaGreen qRT-PCR kit for GAPDH
amplification.
ExCellenCT One-Step EvaGreen/TaqProbe
qRT-PCR Kit
Designed for quantitative real-time analysis of miRNA samples,
abm’s ExCellenCT Lysis Kit prvides an unique and effective alterna-
abm’s EvaGreen miRNA qPCR MasterMix offers superb perfor-
tive method to extract and process RNA templates directly from
mance in sensitivity, signal-to-noise ratio, and reduction of non-spe-
cultured cells in less than 15 minutes. proprietary qRT-PCR Enzyme
cific PCR amplication. The proprietary formulation of our Master-
Mix contains stabilizers and enhancers to optimize the two reactions
Mix is optimized for the amplification of cDNA generated from
in a real-time "single step". Coupled together, this complete system
abm’s miRNA cDNA synthesis kits. Available with either ROX or
provides the ultimate convenience in generating consistent,
fluorescein as the internal passive reference dye, EvaGreen miRNA
reproducible, and accurate results from 10-105 of cultured cells.
qPCR MasterMixes are compatible with a range of qPCR machines.
Cell Lysis

Incubate for
8 min.
RT from Lysates
RT-PCR Analysis

Add stop solution
incubate 2 min.
qPCR Control Template Kit
abm’s qPCR Control Template Kit is a complete system including a
RNA
5’
human RNA template, cDNA template and a β-actin primer mix,
3’
5’
3’
RT Primer
allowing the user to easily incorporate a reliable positive control into
abm’s ExCellenCT Lysis Kit
5X All-In-One RT MasterMix (with ExCellenCT Lysys Kit)
EvaGreen miRNA qPCR MasterMixes
EvaGreen 2X qPCR MasterMix and TaqProbe 2X qPCR MasterMix
ExCellenCT One-Step EvaGreen qRT-PCR Kit and ExCellenCT One-Step TaqProbe qRT-PCR Kit
Two-Step RT-PCR Kit
their existing qPCR setups. The kit can also be used as a convenient
One-Step RT-PCR Kit
tool for demonstration purposes within teaching laboratories
Next Generation Quantitative PCR
qPCR Lentivirus Titration (Titer) Kit
qPCR Adeno-Associated Virus Titration Kit
Designed with titer primers for all HIV-1 based vector detection,
The qPCR Adeno-Associated Virus Titration (Titer) Kit provides a
abm’s Lentivirus Titration Kit enables fast and simple lentivirus
fast and simple way to perform Adeno-Associated Virus titration.
titration. Its rapid RNA extraction method and qRT-PCR based viral
abm’s kit has demonstrated itself to be superior in both sensitivity
RNA quantification allow for assay completion in just 2 hours. Along
and specificity compared to similar products on the market, while
with our online titer calculator, titration of lentiviral preparations has
keeping non-specific background signals to a minimum.
never been easier.
• No RNA purification - saves time and eliminates inaccuracies
• No RNA purification - saves time and eliminates inaccuracies
• Ready-to-use reagent mix - reduces variability
• Ready-to-use reagent mix- reduces variability
• qRT-PCR in one step - more sensitive and accurate than other
• Rapid set-up, qPCR assay can be run in under 2 hours
methods
• No NTC amplification - the only one on the market that eliminates
NTC signal due to our unique primer design.
abm’s qPCR Product List
Cat. No
MasterMix-R, LR, iC, S
Product Name
Size
EvaGreen 2X qPCR MasterMix
500 Rxn (20 μl), 4 x 1.25 ml
EvaGreen 2X qPCR MasterMix
2000 Rxn (20 μl), 16 x 1.25 ml
MasterMix-5R, 5L, 5C, 5S
EvaGreen 5X qPCR MasterMix
1000 Rxn (20 μl), 4 x 1.0 ml
MasterMix-ER, EL, EC, ES
EvaGreen Express 2X qPCR MasterMix
500 Rxn (20 μl), 4 x 1.25 ml
G471-R, LR, iC, S
One-Step EvaGreen qRT-PCR Kit
100 Rxn (20 μl/rxn)
G917-R, LR, iC, S
ExCellenCT One-Step EvaGreen qRT-PCR Kit
25 Preps, 100 x 20 μl Rxn
MasterMix-P, PL, PC, PS, PM
TaqProbe 2X qPCR MasterMix
500 Rxn (20 μl), 4 x 1.25 ml
G493-P, PL, PC, PS
One-Step TaqProbe qRT-PCR Kit
100 Rxn (20 μl/rxn)
G918-P, PL, PC, PS
ExCellenCT One-Step TaqProbe qRT-PCR Kit
25 Preps, 100 x 20 μl Rxn
G929
qPCR Control Template Kit
100 Rxn
MasterMix-mR, mL, mC, mS
EvaGreen miRNA qPCR MasterMix
500 Rxn (20 μl), 4 x 1.25 ml
LV900
qPCR Lentivirus Titration(Titer) Kit
100 Rxn
G931
qPCR Adeno-Associated Virus Titration (Titer) Kit
100 Rxn
MasterMix-R-XL, LR-XL, iC-XL, S-XL
R : ROX, L : Low ROX, C : Fluorescein, S : No-dye
Applied Biological Materials Inc.
#1-3671 Viking Way Richmond, BC
V6V 2J5 CANADA
www.abmgood.com
T : 02 3462 8658
www.itsbio.co.kr
F : 02 3462 8659
[email protected]