Simply greater performance for all your qPCR needs€¦ · copies ampliﬁed in four replicate...

USB® VeriQuestTM qPCR Master Mixes

Greater sensitivity – Reliable amplification of low expressing targets for accurate expression analysis High efficiency PCR across the full dynamic range

Greater specificity – Enzymology expertise for specificity in target amplification and carry-over contamination prevention Accurate target quantitation with prevention of non-specific amplification

Greater confidence – Consistent and reproducible results, even from challenging templates Robust results on a variety of templates

Greater performance – Optimized formulation for quality data analysis Pre-assembled reactions that remain stable for 72 hours at room temperature

Simply greater performance for all your qPCR needs

> SENSITIVITY > SPECIFICITY > CONFIDENCE > PERFORMANCE

USB VeriQuest qPCR Master MixesGreater qPCR performance from a trusted source

> RESULTS

VeriQuest™ Real-Time qPCR Master MixesUSB® VeriQuest qPCR Master Mixes are supplied as 2X pre-mixed formulations containing both Passive Reference Dye and Uracil-DNA Glycosylase (UDG) in an optimized buffer for quality results in real-time quantitative PCR (qPCR) assays. The proprietary reaction buffer and the hot start polymerase enhance qPCR amplification by reducing primer-dimer formation and non-template priming, increasing specificity and sensitivity.

USB VeriQuest qPCR Master Mixes provide reproducible, consistent results while maintaining precision and efficiency. Consistency over a broad dynamic template concentration range allows 7 orders of magnitude linear detection for our SYBR Green formulation and 9 orders of magnitude linear detection for probe formulation (Figure 1).

Single-tube, TaqMan® Probe and SYBR Green master mix formulations available for qPCR

Exceptional, robust performance even with challenging GC-rich regions (Figure 2)

Highly stable qPCR master mix can remain at room temperature for 72 hours in a pre-assembled reaction

Enhanced amplification sensitivity and accuracy of low copy number detection (Figure 3)

Carry-over contamination prevention with UDG (Figure 4) Outstanding reproducibility across 96-wells (Figure 5) One-tube master mix — just add template, primers, and water

Figure 1. VeriQuest Probe qPCR Master Mix linear detection range

Consistent results allowing 9 orders of magnitude linear detection.Amplification plot from real-time PCR for a dilution series of a GAPDH synthetic target with starting amounts of 1010 copies amplified in four replicate reactions using the ABI™ 7500 PCR System and GAPDH primer-probe set (FAM-BHQ®-1).

Fig. 1. Linear detection range of VeriQuest Probe qPCR Master Mix. Real-time amplification plot from a 10-fold dilution series of a GAPDH synthetic target with starting amounts of 1010 copies amplified in four replicate reactions using the ABI 7500 PCR System and GAPDH primer-probe set (FAM-BHQ®-1).

Figure 3. VeriQuest Probe qPCR Master Mix sensitivity of limited target detection

Simultaneous detection of both low and high expression targets. Mean Ct values (top) and standard curve (bottom) from real-time PCR for a dilution series of human genomic DNA. The single copy gene, RNaseP, was amplified from 6.6-66 pg human gDNA, which corresponds to ~2-20 copies of target gene.

Fig. 3. VeriQuest Probe qPCR Master Mix sensitivity of limited target detection

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20 10 4 3 2

Ct

input copies

R² = 0.9924

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Ct

log copy number

Simultaneous detection of both low and high expres-sion targets. Mean Ct values (left) and standard curve (right) from real-time PCR for a dilution series of human genomic DNA. The single copy gene RNaseP, was amplified from 6.6-66 pg human gDNA, which corresponds to ~2-20 copies of target gene.Fig. 3. VeriQuest Probe qPCR Master Mix sensitivity of limited target detection

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20 10 4 3 2

Ct

input copies

R² = 0.9924

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log copy number

Simultaneous detection of both low and high expres-sion targets. Mean Ct values (left) and standard curve (right) from real-time PCR for a dilution series of human genomic DNA. The single copy gene RNaseP, was amplified from 6.6-66 pg human gDNA, which corresponds to ~2-20 copies of target gene.

Fig. 2. GC-rich target e�ciently amplied with VeriQuest Probe qPCR Master Mix

Ampli�cation plots (left) and mean Ct values (right) from real–time PCR for 10 ng (left), 1 ng, 100 pg and 10 pg (right) human male genomic DNA ampli�ed in 4 replicate reactions with VeriQuest Probe qPCR Master Mix (USB VQ), ABI TaqMan ® Gene Expression Master Mix (ABI GE) and ABI TaqMan Universal Master Mix II (ABI UII).

USB VQ

ABI U II

ABI GE

10 ng gDNAPROC

USB VeriQuest Probe ABI Gene Expression ABI Universal II

10 ng 24.6 28.5 24.0

1 ng 28.0 32.7 27.7

100 pg 31.4 36.9 31.7

10 pg 34.8 40.7 35.9

20 25 30 35 40 45

Ct

Figure 2. GC-rich target efficiently amplified with VeriQuest Probe qPCR Master Mix

Amplification plots (top) and mean Ct values (bottom) from real-time PCR for 10 ng (top and bottom), 1 ng, 100 pg, and 10 pg (bottom) human male genomic DNA amplified in 4 replicate reactions with VeriQuest Probe qPCR Master Mix (USB VQ), ABI TaqMan Gene Expression Master Mix (ABI GE), and ABI TaqMan Universal Master Mix II (ABI U II).

Fig. 2. GC-rich target e�ciently amplied with VeriQuest Probe qPCR Master Mix

Ampli�cation plots (left) and mean Ct values (right) from real–time PCR for 10 ng (left), 1 ng, 100 pg and 10 pg (right) human male genomic DNA ampli�ed in 4 replicate reactions with VeriQuest Probe qPCR Master Mix (USB VQ), ABI TaqMan ® Gene Expression Master Mix (ABI GE) and ABI TaqMan Universal Master Mix II (ABI UII).

USB VQ

ABI U II

ABI GE

10 ng gDNAPROC

USB VeriQuest Probe ABI Gene Expression ABI Universal II

10 ng 24.6 28.5 24.0

1 ng 28.0 32.7 27.7

100 pg 31.4 36.9 31.7

10 pg 34.8 40.7 35.9

20 25 30 35 40 45

Ct

Fig. 5. VeriQuest SYBR Green qPCR Master Mix delivers exceptional reproducibility across a 96-well plate.

Ampli�cation plots and melting curves from Real-time PCR of 100 pg of cDNAs reverse-transcribed from HeLa total RNA ampli�ed in 96 repli -cate reactions with VeriQuest SYBR Green qPCR Master Mix for GAPDH detection.

aver stdev CVCt 23.6 0.05 0.22%ΔRn 9.66 0.20 2.11%







Figure 4. VeriQuest™ SYBR® Green qPCR Master Mix carry-over contamination efficiency

UDG eliminates at least 106 copies of dUTP-containing templates without affecting PCR performance. E. coli UDG removal of 106 copies of dUTP-containing GAPDH amplicon (top) and no loss in assay sensitivity from real-time PCR for Chromosome Y genomic marker from 2 of human male genomic DNA (bottom).

Fig. 4. VeriQuest SYBR Green qPCR Master Mix carry-over contamination efficiency

UDG eliminates at least 106 cop -ies of dUTP-containing templates without affecting PCR perfor-mance. E. coli UDG removal of 106 copies of dUTP-containing GAPDH amplicon (left) and no loss in assay sensitivity from real-time PCR for Chromosome Y genomic marker from 2 of human male genomic DNA (right).

Amplification of dUTP templates Amplification of chromosome Y marker from genomic DNA

VeriQuest SYBR Green qPCR Master Mix with UDGwithout UDGNo Template Control (NTC)

VeriQuest SYBR Green qPCR Master Mix with UDGwithout UDG

8

6

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2

0

RFU

0 10 20 30 40

Cycle Number

8

6

4

2

0

RFU

0 10 20 30 40

Cycle Number

2 ng human gDNAAvg. Ct=29.02 ± 0.09Avg. Ct=28.97 ± 0.14

Fig. 4. VeriQuest SYBR Green qPCR Master Mix carry-over contamination efficiency

UDG eliminates at least 106 cop -ies of dUTP-containing templates without affecting PCR perfor-mance. E. coli UDG removal of 106 copies of dUTP-containing GAPDH amplicon (left) and no loss in assay sensitivity from real-time PCR for Chromosome Y genomic marker from 2 of human male genomic DNA (right).

Amplification of dUTP templates Amplification of chromosome Y marker from genomic DNA

VeriQuest SYBR Green qPCR Master Mix with UDGwithout UDGNo Template Control (NTC)

VeriQuest SYBR Green qPCR Master Mix with UDGwithout UDG

8

6

4

2

0

RFU

0 10 20 30 40

Cycle Number

8

6

4

2

0

RFU

0 10 20 30 40

Cycle Number

2 ng human gDNAAvg. Ct=29.02 ± 0.09Avg. Ct=28.97 ± 0.14

Figure 5. VeriQuest SYBR Green qPCR Master Mix delivers exceptional reproducibility across a 96-well plate

VeriQuest Standard Mode qPCR Master Mixes – SYBR Reactions based on 50 μl total volume

VeriQuest SYBR Green qPCR Master Mix (2X) One-tube master mix for real-time PCR, including SYBR Green, UDG, and ROX™ Passive Reference Dye

75600 40 reactions 1 ml 200 reactions 5 ml 400 reactions 2 x 5 ml 1,000 reactions 5 x 5 ml 2,000 reactions 10 x 5 ml

VeriQuest SYBR Green qPCR Master Mix with Fluorescein (2X) One-tube master mix for real-time PCR, including SYBR Green, UDG, and Fluorescein Passive Reference Dye


VeriQuest Standard Mode qPCR Master Mixes – Probe Reactions based on 50 μl total volume

VeriQuest Probe qPCR Master Mix (2X) One-tube master mix containing UDG and ROX Passive Reference Dye


VeriQuest Probe qPCR Master Mix, No Reference Dye (2X) One-tube master mix containing UDG for instruments that do not require a Passive Reference Dye


Amplification plots and melting curves from real-time PCR of 100 pg of cDNAs reverse-transcribed from HeLa total RNA amplified in 96 replicate reactions with VeriQuest SYBR Green qPCR Master Mix for GAPDH detection.

The introduction of commercial microarrays by Affymetrix in the mid-1990s revolutionized gene expression analysis by offering previously inconceivable quality and consistency with unmatched depth and breadth of genomic coverage. Simultaneously, real-time quantitative PCR emerged as a complementary technology to identify and measure differential gene expression with the benefits of specificity, sensitivity, and broad dynamic range(1). The joining together in one company, Affymetrix—innovators in gene expression, and USB—quality specialists in enzymology and reagent formulation, led to the introduction of the USB® VeriQuest™ qPCR Master Mixes.

There are many methods available for microarray validation and Affymetrix offers multiple options including VeriQuest qPCR master mixes and branched DNA base QuantiGene® products. While quantitative PCR is a well-accepted method for validating array data as shown in the influential MicroArray Quality Control (MAQC) study(2), having a highly specific and sensitive master mix with a wide dynamic range is key for precise quantification and validation. With the benefits of the VeriQuest master mixes, customers can be certain this optimized formulation will meet their microarray validation needs.

With exceptional sensitivity, specificity, and dynamic range, VeriQuest master mixes are useful tools for microarray validation (2, 3)

Accurately quantify differences in mRNA expression and genotyping analysis Confirm differential gene expression from array analyses Measure abundance of DNA or RNA sequences in clinical or industrial samples

VeriQuest real-time PCR master mixes are the fast, easy, familiar, and cost-effective validation solutions

qPCR is highly sensitive Small sample volumes are effective Quickly generate validation data with either fast or standard mode cycling Familiar technology using readily available laboratory equipment

References

1. VanGuilder, H. D., Vrana, K. E., and Freeman, W. M. (2008) Biotechniques 44 (5), 619-626.

2. MAQC Consortium (2010) Nature Biotechnology 28, 827-838.

3. Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, 125–128.

Revolution driving innovation

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For efficient sample quantification of both high and low expressing targets

Detect as few as two copies of your target High efficiency PCR across the full

dynamic range Optimal performance creates sensitive results

Accurate quantitation of your target without amplification of non-specific primers

Formulated to prevent non-specific amplification Prevent carry-over contamination with UDG Enzymology expertise creates specific results

Reproducible and robust results on a variety of templates

Robust results on challenging templates Single tube reduces pipetting errors Consistency creates confidence in your results

Optimized master mix formulation for quality data analysis

Pre-assembled reactions stable for 72 hours Reference dyes matched to every instrument Optimal formulation helps your performance

Simply greater qPCR performance means results you can trust.

>

SENSITIVITY

>

SPECIFICITY

>

CONFIDENCE

>

PERFORMANCE

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VeriQuest™ Fast qPCR Master MixesUSB® VeriQuest Fast qPCR Master Mixes dramatically reduce your run time without sacrificing sensitivity and quality. The master mixes offer higher specificity and sensitivity, virtually eliminating non-specific primer amplification which can negatively affect the efficiency and accuracy of the data (Figure 6). The consistency and specificity afforded by the VeriQuest fast master mixes make them ideally suited for gene expression studies and microarray validation.

Optimized for fast mode thermal cycling conditions for results in one-third of the time

Reproducibility and consistency over a broad dynamic range: 8 orders of magnitude linear detection range (Figures 6 and 7)

Exceptional, robust performance even with challenging GC-rich regions

Sensitivity and precision with limited targets (Figure 8) Highly stable master mix can remain at room temperature for 72

hours in a pre-assembled reaction Contains dUTP and Uracil-DNA Glycosylase (UDG) for carry-over

contamination prevention Exceptional results with performance comparisons (Figure 9)

Fig. 6. Linear detection range

Linear detection range of VeriQuest Fast Probe qPCR Master Mix. Amplifi-cation plot and standard curve from real-time PCR for a dilution series of a synthetic target with starting amounts of 1010 copies amplified in four repli-cate reactions using the ABI 7500 Real-Time PCR System and GAPDH prim-ers. Reaction performed using fast mode cycling with 5 minutes activation at 95°C. The amplification process was linear over eight orders of magnitude.

Figure 6. VeriQuest Fast Probe qPCR Master Mix linear detection range

Consistent results allowing 8 orders of magnitude linear detection of VeriQuest Fast Probe qPCR Master Mix. Amplification plot and standard curve from real-time PCR for a dilution series of a synthetic target with starting amounts of 1010 copies amplified in four replicate reactions using the ABI™ 7500 Real-Time PCR System and GAPDH primers. Reactions were performed using fast mode cycling with 5 minutes activation at 95°C.

Figure 8. High sensitivity and precision in limited target quantification with VeriQuest Fast Probe qPCR master mix

Amplification plot (top) and standard curve (bottom) from real-time PCR for a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cDNAs reverse-transcribed from HeLa total RNA.

Fig. 7. High sensitivity and precision

High sensitivity and precision in limited target quantification. Amplification plot (bottom) and standard curve (top) from real-time PCR for a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cDNA reverse-transcribed from HeLa total RNA.

Cyclophilin

10 4 3 2 1 ng RNA

Kras

10 4 3 2 1 ng RNA

Cyclophiliny = -3.3841x + 24.142

EPCR = 97.5%R² = 0.9977

Krasy = -3.3502x + 31.829

EPCR = 98.8%R² = 0.9973

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-0.2 0 0.2 0.4 0.6 0.8 1 1.2

Ct

log input

Cyclophilin Kras

Fig. 7. High sensitivity and precision

High sensitivity and precision in limited target quantification. Amplification plot (bottom) and standard curve (top) from real-time PCR for a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cDNA reverse-transcribed from HeLa total RNA.

Cyclophilin

10 4 3 2 1 ng RNA

Kras

10 4 3 2 1 ng RNA

Cyclophiliny = -3.3841x + 24.142

EPCR = 97.5%R² = 0.9977

Krasy = -3.3502x + 31.829

EPCR = 98.8%R² = 0.9973

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-0.2 0 0.2 0.4 0.6 0.8 1 1.2

Ct

log input

Cyclophilin Kras

Figure 7. VeriQuest Fast SYBR Green qPCR Master Mix linear detection range

Consistent results allowing 8 orders of magnitude linear detection. Amplification plot and standard curve from real-time PCR for a dilution series of a synthetic target with starting amounts of 109 copies amplified in four replicate reactions using the ABI 7500 Real-Time PCR System and GAPDH primers in fast mode with 5 minutes activation at 95°C.

Revolution driving innovation

Figure 9. Exceptional results with performance comparison

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-4.0 -2.0 0.0 2.0

Ct

log ng input

B2M

ABI™ Fast SYBR

Bio -Rad iTaq™ Fast

VeriQuest™ Fast SYBR®

VeriQuest Fast SYBRy = -3.1802 + 23.205EPCR = 106.3 %R2 = 0.9999

Bi o-Rad iTaq Fast SYBRy = -3.3764 + 22.908EPCR = 97.8 %R2 = 0.9991

ABI Fast SYBRy = -3.1984 + 23.75 5EPCR = 105.4 %R2 = 0.9995

Fig. 8. Exceptional results with performance comparison

Standard curves from real-time PCR for a 10-fold dilution series of 10 ng to 1 pg cDNA reverse-transcribed from HeLa total RNA ampli�ed in duplicate reactions with VeriQuest Fast SYBR Green qPCR Master Mix, Bio-Rad iTaq Fast SYBR Green Supermix with ROX™, and ABI Fast SYBR Green Master Mix.

VeriQuest Fast qPCR Master Mixes – SYBR Reactions based on 20 μl total volume

VeriQuest Fast SYBR Green qPCR Master Mix (2X)A 2X pre-mixed formulation of SYBR Green qPCR master mix designed for fast mode cycling conditions

75690 100 reactions 1 ml 500 reactions 5 ml 1,000 reactions 2 x 5 ml 2,500 reactions 5 x 5 ml 5,000 reactions 10 x 5 ml

VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein (2X)2X pre-mixed formulation of SYBR Green qPCR master mix with Fluorescein, designed for fast mode cycling conditions


Standard curves from real-time PCR for a 10-fold dilution series of 10 ng to 1 pg cDNA reverse-transcribed from HeLa total RNA amplified in duplicate reactions with VeriQuest Fast SYBR Green qPCR Master Mix, Bio-Rad iTaq Fast SYBR Green Supermix with ROX, and ABI Fast SYBR Green Master Mix. Reactions were performed using fast mode cycling conditions with a 5 minute activation at 95˚ C.

VeriQuest Fast qPCR Master Mixes – Probe Reactions based on 20 μl total volume

VeriQuest Fast Probe qPCR Master Mix (2X)2X pre-mixed probe-based qPCR master mix containing ROX, designed for fast mode cycling conditions


VeriQuest Fast Probe qPCR Master Mix, No Reference Dye (2X)A 2X pre-mixed formulation of probe-based qPCR master mix containing UDG


VeriQuestTM One-Step qRT-PCR Master MixesUSB® VeriQuest One-Step qRT-PCR Master Mixes are ready-to-usemaster mixes for RNA quantification. The optimized buffer formulation offers specificity, robustness, and reliability for reverse transcription and qPCR detection (Figures 10 and 11). USB VeriQuest One-Step qRT-PCR Master Mixes provide maximum convenience for a real-time, quantitative analysis of RNA templates in a single-reaction format. The RT-PCR process converts and amplifies single-stranded RNA template yielding double-stranded DNA product.

This kit exhibits excellent sensitivity as it can detect fewer than 10 target copies, performs over a broad, linear dynamic range of 7 orders of magnitude (Figures 10 and 11), and is compatible with all real-time PCR instruments.

One-step, sequential reaction format for minimum hands-on time Exceptional stability even after 10 freeze-thaw cycles (Figure 12) Premium results with product performance comparisons (Figure 13) Easily perform analysis of single or multiplex targets from multiple

RNA samples (Figure 14) Pre-mixed format with VeriQuest qRT-PCR mixes reduces potential

contamination Multiple platform compatibility with all real-time master mixes

Figure 10. Consistent results over an extremely broad dynamic range with VeriQuest SYBR Green One-Step qRT-PCR Master Mix

Linear detection range of VeriQuest SYBR Green One-Step qRT-PCR Master Mix. RNA from human muscle cells was reverse transcribed and amplified using 18S primers. Triplicate reactions were run on an ABI™ 7500 Real-Time PCR System.

Figure 11. Consistent results over an extremely broad dynamic range with VeriQuest Probe One-Step qRT-PCR Master Mix

Linear detection range of VeriQuest Probe One-Step qRT-PCR Master Mix. RNA from human muscle cells was reverse transcribed and amplified using 18S primers. Triplicate reactions were run on an ABI 7500 Real-Time PCR System.

Figure 12. Freeze-thaw stability with VeriQuest Probe One-Step qRT-PCR Master Mix

No loss in performance after 10 freeze-thaw cycles. Amplification plot from real-time PCR for a 10-fold dilution series of 100 ng HeLa total RNA. The master mix was subjected to 0, 3, and 10 freeze-thaw cycles.

Fig. 12. Freeze-thaw stability with VeriQuest Probe One-Step qRT-PCR Master Mix

No loss in performance after 10 freeze-thaw cycles. Ampli�cation plot from real-time PCR for a 10-fold dilution series of 100 ng HeLa total RNA. The master mix was subjected to 0, 3, and 10 freeze-thaw cycles.

# fr eeze-thaw cycles

0 3 10

slope -3.4337 -3.4068 -3.4659

EPCR 95.54% 96.58% 94.32%

R2 0.9999 0.9999 0.9998

Fig. 12. Freeze-thaw stability with VeriQuest Probe One-Step qRT-PCR Master Mix

No loss in performance after 10 freeze-thaw cycles. Ampli�cation plot from real-time PCR for a 10-fold dilution series of 100 ng HeLa total RNA. The master mix was subjected to 0, 3, and 10 freeze-thaw cycles.

# fr eeze-thaw cycles

0 3 10

slope -3.4337 -3.4068 -3.4659

EPCR 95.54% 96.58% 94.32%

R2 0.9999 0.9999 0.9998

VeriQuest Real-Time Reverse Transcription Master Mixes – SYBR Reactions based on 50 μl total volume

VeriQuest SYBR Green One-Step qRT-PCR Master Mix (2X) One-step qRT-PCR master mix that includes SYBR Green and ROX Passive Reference Dye for use on all instrument platforms that utilize ROX™

75705 40 reactions 1 ml 200 reactions 5 ml

VeriQuest SYBR Green One-Step qRT-PCR Master Mix with Fluorescein (2X) One-step qRT-PCR master mix that includes SYBR Green and Fluorescein Passive Reference Dye for use on all instrument platforms that utilize fluorescein


Figure 13. Exceptional results with performance comparisonsFig. 13. Exceptional results with performance comparisons

slope EPCR R2

ABI TaqMan Fast Virus 1-Step -3.4087 96.50% 0.9994

ABI TaqMan RNA-to-CT 1-Step -3.2142 104.70% 0.9953

VeriQuest One-Step Probe -3.4057 96.62% 0.9998

13.0

19.8 23.4

26.8

30.4 33.2

14.0

20.9

24.4 27.8

30.7 32.9

13.3

20.0

23.6 26.9

30.5 33.5

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100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg

Ct

HeLa Total RNA

ABI TaqMan Fast Virus 1-Step

ABI TaqMan RNA-to-CT™ 1-Step

VeriQuest One-Step Probe

16.5 17.5 16.6

Fig. 13. Exceptional results with performance comparisons

slope EPCR R2

ABI TaqMan Fast Virus 1-Step -3.4087 96.50% 0.9994

ABI TaqMan RNA-to-CT 1-Step -3.2142 104.70% 0.9953

VeriQuest One-Step Probe -3.4057 96.62% 0.9998

13.0

19.8 23.4

26.8

30.4 33.2

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20.9

24.4 27.8

30.7 32.9

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30.5 33.5

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100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg

Ct

HeLa Total RNA

ABI TaqMan Fast Virus 1-Step

ABI TaqMan RNA-to-CT™ 1-Step

VeriQuest One-Step Probe

16.5 17.5 16.6

VeriQuest Real-Time Reverse Transcription Master Mixes – Probe Reactions based on 50 μl total volume

VeriQuest Probe One-Step qRT-PCR Master Mix (2X) One-tube, one-step qRT-PCR master mix that includes ROX Passive Reference Dye for use on all instrument platforms that utilize ROX


VeriQuest Probe One-Step qRT-PCR Master Mix, No Reference Dye (2X) One-tube, one-step qRT-PCR master mix for RNA quantitation without a passive reference dye for use on all instrument platforms that do not require reference dye


Figure 14. Consistent results with single, duplex, or triplex target detection with VeriQuest™ Probe qRT-PCR Master Mix

Standard curves from real-time PCR for a 10-fold dilution series of 100 ng to 10 pg of HeLa total RNA amplified in duplicate reactions using the ABI 7500 Fast Real-Time PCR System. Actin Vic-MGB, Cyclophilin Cy3-BHQ-1 probe and EEF1G Fam-BHQ-1 probe were used.

Fig. 14. Consistent results with single, duplex, or triplex target detection with VeriQuest Probe qRT-PCR Master Mix.

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Ct

log ng RNA input

1-plex Actin

2-plex Actin (EEF1G)

2-plex Actin (Cyclophilin)

2-plex Actin (GAPDH)

3-plex Actin (EEF1G, Cyclophilin)

EPCR R2 EPCR R2 EPCR R2

1-plex Actin 94% 0.99991-plex EEF1G 98% 0.99971-plex Cyclo 99% 0.99991-plex GAPDH 99% 0.99992-plex Actin EEF1G 94% 0.9999 97% 0.99952-plex Actin Cyclophilin 97% 0.9997 95% 0.99952-plex Actin GAPDH 95% 0.9998 102% 0.99993-plex Actin EEF1G Cyclophilin 102% 0.9997 100% 0.9999 97% 0.9989

100 ng 10 pg 100 ng 10 pg 100 ng 10 pg2-plex Actin EEF1G 0.0 0.1 0.1 0.22-plex Actin Cyclophilin -0.3 -0.3 0.2 -0.22-plex Actin GAPDH 0.2 0.0 0.1 0.03-plex Actin EEF1G Cyclophilin -0.3 -0.2 0.4 0.3 0.2 -0.2

Target 1 Target 2 Target 3Ct Difference Compared to 1 Plex

Target 1 Target 2 Target 3

Target 3Target 1 Target 2 Target 3

Target 1 Target 2

Standard curves from real-time PCR for a 10-fold dilution series of 100 ng to 10 pg of HeLa total RNA amplified in duplicate reactions using the ABI 7500 Fast Real-Time PCR System. Actin Vic-MGB, Cyclophilin Cy3-BHQ-1 probe and EEF1G Fam-BHQ-1 probe were used.

Fig. 14. Consistent results with single, duplex, or triplex target detection with VeriQuest Probe qRT-PCR Master Mix.

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-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5

Ct

log ng RNA input

1-plex Actin

2-plex Actin (EEF1G)

2-plex Actin (Cyclophilin)

2-plex Actin (GAPDH)

3-plex Actin (EEF1G, Cyclophilin)

EPCR R2 EPCR R2 EPCR R2

1-plex Actin 94% 0.99991-plex EEF1G 98% 0.99971-plex Cyclo 99% 0.99991-plex GAPDH 99% 0.99992-plex Actin EEF1G 94% 0.9999 97% 0.99952-plex Actin Cyclophilin 97% 0.9997 95% 0.99952-plex Actin GAPDH 95% 0.9998 102% 0.99993-plex Actin EEF1G Cyclophilin 102% 0.9997 100% 0.9999 97% 0.9989

100 ng 10 pg 100 ng 10 pg 100 ng 10 pg2-plex Actin EEF1G 0.0 0.1 0.1 0.22-plex Actin Cyclophilin -0.3 -0.3 0.2 -0.22-plex Actin GAPDH 0.2 0.0 0.1 0.03-plex Actin EEF1G Cyclophilin -0.3 -0.2 0.4 0.3 0.2 -0.2

Target 1 Target 2 Target 3Ct Difference Compared to 1 Plex

Target 1 Target 2 Target 3

Target 3Target 1 Target 2 Target 3

Target 1 Target 2

Standard curves from real-time PCR for a 10-fold dilution series of 100 ng to 10 pg of HeLa total RNA amplified in duplicate reactions using the ABI 7500 Fast Real-Time PCR System. Actin Vic-MGB, Cyclophilin Cy3-BHQ-1 probe, and EEF1G Fam-BHQ-1 probe were used.

Product comparison - VeriQuest Probe One-Step qRT-PCR Master Mix. Ct values, R2, and PCR efficiency from real-time PCR for a 10-fold dilution series of 100 ng to 100 fg HeLa total RNA amplified in duplicate reactions with VeriQuest Probe One-Step qRT-PCR Master Mix, ABI™ TaqMan® Fast Virus 1-Step Master Mix, and ABI TaqMan RNA-to-CT™ 1-Step Kit using the ABI 7500 Fast Real-Time PCR System and GAPDH primers and probe (Fam-BHQ®) in standard mode with 15 minute RT reaction at 50°C.

usb.affymetrix.com Please visit our website for international distributor contact information.“For Research Use Only. Not for use in diagnostic procedures.”

© 2011 Affymetrix Inc. All rights reserved.

Affymetrix, USB, ExoSAP-IT, PrepEase, and QuantiGene are registered trademarks of Affymetrix, Inc. VeriQuest is a trademark of Affymetrix, Inc. ExoSAP-IT is covered by US Patent Nos. 6,379,940 and 6,387,634.

PrepEase products are covered under European Patent EP 0496822 and US Patent 6,428,703. Taq DNA Polymerase—sold under licensing arrangements with Applied Biosystems. Purchase is accompanied by a

limited license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee,

either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. SYBR is a registered trademark of Molecular Probes, Inc. and is provided under an agreement with Molecular Probes, Inc.

ABI is a trademark of Life Technologies. iTaq is a trademark of Bio-Rad. TaqMan is a registered trademark of Roche Molecular Systems, Inc. RNA-to-CT is a trademark of Roche Molecular Systems, Inc. ROX is a

trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. BHQ-1 is a registered trademark of Biosearch Technologies. All trademarks are the property of their respective owners.

DNA isolation and purification

Chromatin Immunoprecipitation (ChIP) Assay Kit

78460 25 ChIP Assays

PrepEase Genomic DNA Isolation Kit

78850 50 preps 78855 250 preps

PrepEase MiniSpin Plasmid Kit

78737 250 preps

PrepEase Plasmid 96-well Plate Kit

78751 4 x 96-well plates 78752 24 x 96-well plates

PrepEase BAC Purification Kit

78722 10 preps

PrepEase Yeast Plasmid Isolation Kit

79220 50 preps

Related products

PCR tools

ExoSAP-IT® PCR Product Cleanup

78250 20 reactions 78200 100 reactions 78201 500 reactions 78202 2,000 reactions 78205 5,000 reactions

HT ExoSAP-IT High-Throughput PCR Product Cleanup

78395 480 reactions x 8-tube strip 5,760 reactions x 1 plate

(12 x 8-tube strips) 23,040 reactions - 4 plates 1,000 reactions (2 ml) 5,000 reactions (10 ml)

PrepEase® DNA Cleanup Kit

78758 50 preps 78759 250 preps

PrepEase PCR Purification 96-well Plates (Ultrafiltration)

78761 10 x 96-well plates 78762 50 x 96-well plates

First-Strand cDNA Synthesis Kit for Real-Time PCR

75780 50 reactions

VeriQuest™ Taq DNA Polymerase

71170 50 units 250 units 1,000 units 5,000 units

Water, Nuclease-Free

71786 10 x 1 ml 100 ml 500 ml 1 L 5 L

RNA isolation and purification

PrepEase RNA Spin Kit

78766 50 preps 78767 250 preps

PrepEase mRNA MiniSpin Kit

78878 12 preps

PrepEase RNA/Protein Spin Kit

78871 50 preps

Isogen Life Science B.V.Veldzigt 2A3454 PW De MeernThe NetherlandsTel: +31 (0)30 688 0771Fax:+31 (0)30 688 8009E-mail: [email protected]

Isogen Life Science N.V./S.A.Stokthoekstraat 10A9140 TemseBelgiumTel: +32 (0)9 351 48 79Fax:+32 (0)9 277 93 03E-mail: [email protected]

Isogen Life Science s.l.u.Paseo del Ferrocarril, 339-bajos 3°Parque Empresarial del Mediterráneo08860 Castelldefels – BarcelonaTel: +34 936 644 894Fax: +34 936 455 089E-mail: [email protected]

Simply greater performance for all your qPCR needs€¦ · copies ampliﬁed in four replicate...

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