brilliant qpcr reagents - agilent · brilliant iii ultra-fast products key product features 1. the...

Brilliant QPCR Reagents

Agilent’s QPCR Reagents

RNA Quantification, 2-StepRNA Quantification, 1-StepDNA (cDNA) Quantification

Probe

Brilliant II QPCR Low ROX MM

Brilliant II QPCR High ROX MM

Brilliant Multiplex MM

Brilliant III Ultra-Fast

QPCR MM

AffinityScript QPCR cDNA Synthesis Kit

Plus:

Brilliant II QRT-PCR Low ROX MM

Brilliant II QRT-PCR High ROX MM

Brilliant Multiplex MM

Brilliant III Ultra-Fast QPCR MM

Brilliant II QPCR Low ROX MM, 1-Step

Brilliant II QPCR High ROX MM, 1-Step

Brilliant III Ultra-Fast QRT-PCR

MM, 1-Step

SYBR®

Green

Brilliant II SYBR® QPCR Low

ROX MM

Brilliant II SYBR® QPCR High

ROX MM


SYBR® QPCR MM

Brilliant II SYBR® Green QRT-PCR MM, 1-

Step

Brilliant II SYBR® QRT-PCR Low ROX

MM, 1-Step

Brilliant II SYBR® QRT-PCR High ROX

MM, 1-Step

Brilliant III Ultra-Fast SYBR®

QRT-PCR MM, 1-Step


Plus:

Brilliant II SYBR® QPCR Low ROX MM

Brilliant II SYBR® QPCR High ROX MM

Brilliant III Ultra-Fast SYBR®

QPCR MM

Detection

Brilliant III Ultra-Fast Products

Key Product Features

1.The enzyme is a Taq mutant which has been engineered by

Agilent specifically for speed

2.

Employs a novel chemical hot start technology which

activates more quickly to save time and minimize non-specific

product formation compared to other hot start methods

Engineering A Faster Mutant of Taq DNA

Polymerase

• Prepare a randomized mutant library of Taq DNA polymerase

• Perform multiple rounds of selection for mutants which excel under fast

conditions

• After screening and rescreening under fast cycling conditions,

mutations are combined to create a library of multi-site mutants

• Fastest mutants selected and characterized for performance in QPCR

Result: A mutant Taq, engineered for fast cycling conditions,

with excellent performance in sensitivity, robustness, and

reproducibility

Faster-Activating Chemical Hot Start

Radioactive incorporation assay was performed at

72°C.

• Most fast qPCR reagents use Taq antibody to inactivate the polymerase at PCR set-

up temperatures

•Antibody hot start is a fast activating method

•Antibody hot start is not as effective as chemical

• Agilent developed a faster-activating chemical hot start by reversibly inactivating

Taq42 with an alternative modification reagent that is more sensitive to heat and pH

changes

Brilliant III Ultra-Fast Shortens Cycling Time by ~60% -

Maintains Efficiency, R2, Dynamic Range, and Sensitivity

Brilliant II Brilliant IIITotal cycling Time: 101 minutes Total cycling time: 40 minutes

(1x 10min@95°C, 40x 30sec@95°C/60sec@60°C) (1x 3min@95°C, 40x 5sec@95°C/10sec@60°C)

R2: 0.997; Eff:95.4% R2: 0.994; Eff:98.16%

NOTE: B7 target (148bp) was amplified from from 2-fold dilutions of plasmid DNA at concentrations ranging from 1536

to 3 copies/Rxn on ABI StepOnePlus (quadruplicates).

Brilliant III Ultra-Fast QPCR Master Mixes - Superior

Sensitivity at Low Target Concentrations

Amplification plot and standard curve for Brilliant III Ultra-Fast QPCR Master Mix (# 600880) showing 10-fold serial

dilution of 100 ng to 10 fg of cDNA from human total RNA detecting 18S rRNA gene target using “Assay on

Demand” assay.

Efficiency = 89%, R2 = 0.994

Brilliant III Ultra-Fast SYBR Green QPCR Master Mix Exhibits Precise

Detection of 2-fold Differences from 1536 Copies Down to ~3 Copies

Efficiency = 90%, R2 = 0.988

10-fold serial dilution of plasmid from 1x 109 to 10 copies

y = -3.4377x + 39.411R2 = 0.999Eff: 95%

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

avg

Ct

(n=

4)

10X dilution series of plasmid (1x109 - 1x100 copies per reaction)

Brilliant III Ultra-Fast QPCR Master Mix Delivers Superior Sensitivity

and Precision to Detect Target Across 9 Orders of Magnitude

Brilliant III Ultra Fast QPCR Reagents

Universal master mix which delivers equal to better

performance under routine cycling conditions and which

provides superior performance under fast cycling (40

cycles of PCR in 40 minutes) when compared to competitor

reagents

Under fast cycling conditions, Brilliant III will deliver

improved performance:

• Increased reproducibility

• Wider dynamic range

• Earlier Cts

• Shorter run times

Advantages Agilent Provides with Brilliant III

•Superior sensitivity and reproducibility under fast cycling

•Minimizes primer-dimer formation

•Complete solutions:

•Agilent offers platforms, reagents (including QPCR references

and sample preparation products), and Bioanalyzer

•Expert support from Agilent’s Technical Services and product

specialists

•Quality control in production for consistently reliable products

Agilent and IDT Co-Marketing Agreement Improves

Performance – Brilliant Reagents with PrimeTime

Assays

• Improved sensitivity - Lower Ct (Cq) values

• Better efficiency

• Greater flexibility

• More economical than alternatives

Brilliant + PrimeTime vs. Competitor A Across Platforms

93%

97%

94%

98% 98%

95%

100%

96%96%97%

102%

99%

104%

99%

95%

103%

96%

99%

86%

89%90%

91%

95%

93%94%

91% 91%90% 90% 90%

86%

93%

90%91%

97%

91%

50%

60%

70%

80%

90%

100%

110%

18S ACTB B2M GAPDH GUSB HPRT PSG8 TBP Average

Ass

ay E

ffic

ien

cy

Calculated qPCR Efficiency ComparisonIDT and Agilent Products vs. Applied Biosystems Products

7900: IDT + Agilent

Mx: IDT + Agilent

7900: Applied Biosystems

Mx: Applied Biosystems

Calculated qPCR Efficiency Comparison

Agilent + IDT Products vs Competitor A Products

7900: IDT + Agilent

Mx: IDT + Agilent

7900: Competitor A

Mx: Competitor A

Agilent-IDT Product Data Sheet

Data generated in multiple locations to ensure reproducibility across platforms, locations, and targets.


Description

• AffinityScript QPCR cDNA Synthesis Kit is designed to deliver the highest efficiency

conversion of RNA to cDNA and is fully optimized for real-time PCR applications

Benefits

• Fast, highly efficient cDNA synthesis for RT-qPCR

• Streamlined protocol produces cDNA in 15 minutes

• Linear detection from 3 pg to 3 µg total RNA

• Master Mix format saves time, reduces pipetting variability

Brilliant III Customer Testimonial

• Customer tested Brilliant III on the Roche LC480 (5/10 cycling) vs the following:

– Roche FastStart Universal SYBR Green Master Mix

– Roche LightCycler 480 SYBR Green I Master Mix

– Sigma SYBR Green JumpStart Taq Ready Mix

– peqlab KAPA SYBR fast qPCR Mastermix

– Finnzymes DyNAmo Color Flash SYBR Green qPCR kit

– Fermentas Maxima SYBR Green qPCR Master Mix

– BioronSiBir Master Mix

– Plus 4 others…

“With Brilliant III we were able to push the boundaries of our research. As we are working

with rare species of cells in a huge background of other cell types, we can now distinguish

the biological picture more clearly….”

“You might be happy to hear that we now also tested additional master mixes (10 in total so

far) and in terms of Biorad Sso Fast, Promega GoTaq and Sigma JumpStart…Brilliant III is

way superior to all of them.”

Dr. L. – GeneWake GmbH


• Customer tested Brilliant III on the AB StepOne Plus (5/10 cycling) vs:

– AB Universal Fast Master Mix

“A few weeks ago you sent me the new Brilliant III Ultra-Fast Master Mix for testing. I

have done several runs so far and I am really pleased with the results. With my other

master mix my target gene was only detected after cycle 35, and not reliably. With

Brilliant III I now get valid results every time. I would be very interested in purchasing

more!”

Ms. R – Universitätsklinikum Erlangen


• Customer tested Brilliant III on the BioRad Chromo4 (5/15/10 cycling) vs:

– BioRad SsoFast Master Mix

“In my lab we are working on DNA repair processes and commonly use Chromatin

immunoprecipitation followed by real-time quantitative PCR. Given the size of our lab we

frequently run into bottlenecks when it comes to reserving time on our only Biorad

Chromo4 real-time PCR cycler. I was first reluctant to switch to Brilliant III within data

collection. I feared I would not be able to directly compare "superfast" data to my

previous measurements - an obvious deal breaker for me at this stage of my experiments.

Not only did my percent recovery of DNA over input DNA stay amazingly consistent using

Brilliant III compared to my standard protocol, it also cut my machine time by 30%.”

Johanna – reseacher in San Diego biotech

Examples of Agilent’s QPCR Reference Material

QPCR Systems Brochure

Lit Station ID: 5990-3495EN


QPCR/QRT-PCR Master

Mixes – For ABI

StepOnePlus Real-Time PCR

instrument Data Sheet


Generation of Nonspecific

Amplification Using TaqMan

Assays-on-Demand Gene

Expression Products

Data Sheet



QPCR/QRT-PCR Master

Mixes – For BioRad CFX96

Real-Time PCR instrument

Data Sheet


Examples of Agilent’s QPCR Reference Material

Mx Data Sheet LC480 Data Sheet

Protocol Quick Reference Guides:

• StepOnePlus

• 7500, 7900HT

• CFX96

• LC480

• RotorGeneQ

• Mx

For More Information

Visit http://www.genomics.agilent.com

Appendix

Brilliant III Data

Novel Hot Start of Brilliant III Delivers Minimal Primer-Dimer Formation


SYBR Green QPCR

Master Mix

Competitor Q’s

Fast SYBR Green

qPCR

R2: 0.941; Eff: 84%

R2= 0.996 Eff:

90%

10X dilution series of

human gDNA of Numb-1

target (305 bp) – known

to generate primer-

dimers

Competitor T’s SYBR

R2: 0.979; Eff: 95%

Competitor R’s

SYBR

R2: 0.994; Eff: 98%

Novel Hot Start of Brilliant III Delivers Minimal Primer-Dimer Formation

Company T generates earlier Cts, but the efficiency is compromised by formation of these artifacts which can compete

with the specific product.

Company Q

Company R

Company T

Agilent Brilliant III

Non-specific

amplification

products

Non-specific

amplification

products

Non-specific

amplification

products

Greater Degree of Confidence in Gene Expression Data - Novel Hot

Start Technology of Brilliant III Ultra-Fast = Absence of Non-Specific

Secondary Products

Brilliant III Ultra-Fast Delivers Improved Specificity

Brilliant III

Ultra-Fast

QPCR

Master Mix

Competitor A

Fast Master

Mix

Eff.= 94%

Eff.=79%

GUS primers/probe set (Competitor A Assays)

Brilliant III Ultra-Fast Delivers Improved Specificity with

Reduction of Primer-Dimers

Competitor A Brilliant III Ultra-Fast

100 10 1 0.1 0.01 NTC 100 10 1 0.1 0.01 NTC

Primer-

dimer

product

Input amount

Trace of GUS primers/probe set

Brilliant III Ultra-Fast Delivers Improved Specificity

Brilliant III

Ultra-Fast

QPCR

Master Mix

Competitor A

Fast Master

Mix

TBP primers/probe set (Competitor A Assays)

Eff.=85%

Eff.=100%

Brilliant III Ultra-Fast Delivers Improved Specificity with

Reduction of Primer-DimersCompetitor A Fast Brilliant III Ultra-Fast

100 10 1 0.1 0.01 NTC 100 10 1 0.1 0.01 NTC

Primer-

dimer

product

Input amount

Trace of TBP primers/probe set

Brilliant III Ultra-Fast Delivers Sensitivity and

Reproducibility at Low Target Concentrations

Standard curves showing detection

of a 2-fold serial dilution from 1536

copies to 3 copies of plasmid DNA

Comp A Fast

Universal

Brilliant III

Ultra-Fast

Eff.=107%

Eff.=101%

Brilliant III Ultra-Fast Provides Sensitivity of Detection

Cyclophilin AOD gene targets

Brilliant III Ultra-Fast Master Mix generates Ct values

~3.4 cycles earlier than the Competitor E Fast qPCR

Master Mix for the Cyclophilin target – this

represents over a 10-fold difference in detection.

Competitor E Fast qPCR

Eff.= 85%


Eff.= 97%

Brilliant III Ultra-Fast Delivers Improved Reliability

NOTE: Reliability comparison of Brilliant III QPCR Master Mix and Company R master mix on 83 commercially

available gene targets on ABI 7900 HT. Master mixes were run under recommended cycling conditions. Brilliant III

total run time: 43 min. Company R total run time : 99 min.

10

15

20

25

30

35

40

18

s R

NA

18

s R

NA

GA

PD

H

GA

PD

H

SIL

V

PP

IA

B2

M

AC

TB

AF

P

AC

TB

PS

AP

AP

OB

B2

M

IGF

BP

5

KR

T

GU

S

BC

L2

L1

CA

LM

1

CR

LF

1

AD

M

GL

G1

BA

X

HP

RT

KIF

4A

EL

AV

L1

GU

S

SL

C25

A4

GN

A1

3

HP

RT

GL

13

GA

UC

HE

R

CR

EB

BP

OP

TN

AD

CY

3

PO

LS

R2A

TIA

MI

CE

LS

R1

LIP

A

FU

RIN

CD

58

IGF

1R

VE

GF

C

RP

S6

KB

1

DP

YD

EG

FR

MO

SP

D1

PT

GS

2

AB

CD

1

AB

CG

NG

R1

TB

P

PL

CB

3

TB

P

AG

PA

T4

HF

E

TG

FB

R1

TY

MP

ML

PH

AN

XA

9

PK

1A

AT

P6

ITP

R1

FG

F2

BD

KR

B1

EG

F

NT

F3

SP

AR

CL

1

KC

NC

1

CE

ND

1

PO

MC

AQ

P1

CH

GA

CA

CN

A1

B

CC

NA

1

MM

P1

3

AC

TN

2

PS

G8

IL1

7B

SC

N1

A

RY

R1

AN

KR

D2

FG

F1

0

GH

1

Cq

Target Gene/Amplicon length (bp)

Brilliant III

Company R

37.05

21.21

15

20

25

30

35

40

GA

PD

H-1

21

BA

CT

-29

9A

CT

B-1

01

GA

DP

H-1

51

GA

PD

H-8

6P

IA-9

7P

IA-1

01

PG

K-1

54

B2

M-9

8B

2M

-11

4P

GK

-11

8P

GK

-13

4B

2M

-86

SO

D-1

96

AN

XA

-21

2P

IA-1

17

AP

OA

-15

4G

PI-

10

0A

NX

A-1

37

DLK

-27

3C

TS

-19

5H

IF-1

43

AT

IC-1

49

AK

R-1

60

MA

RC

A-1

27

CS

DE

-32

7N

DU

-12

0M

AP

-16

1ID

H-1

12

CT

N-8

6A

RF

-11

1N

UD

T-3

00

TU

B-1

02

FO

X-1

39

HP

RT

-94

LIT

-20

1D

HX

-20

0S

LC

-12

9N

DU

-10

1C

XC

R-1

30

DN

AJ-1

19

DU

SP

-13

7U

SP

-25

7C

CL

-15

3C

AS

P-1

02

EG

R=

76

EG

R-2

01

PD

K-1

15

JU

P-7

9S

NA

-11

9D

US

P-1

02

SP

R-1

87

CD

H-2

58

CU

L-1

06

BA

K-3

07

RF

G-1

39

NF

IL-2

36

EL

FA

-13

5G

RK

-10

3F

SS

-13

6H

IST

-17

8C

D-1

10

NR

-12

5A

VE

N-2

53

PC

D-1

02

BIR

C-3

61

DF

F-1

40

TZ

-11

7A

BC

-20

5A

BC

-15

6M

GS

T-3

26

MB

L-2

87

GR

P-1

01

AB

C-2

82

AL

B-3

50

AL

B-2

90

AL

OX

-36

3U

TS

-14

6E

RV

-25

0M

AP

-38

1A

LB

-35

8D

ID-3

45

OX

R-3

40

KR

T-3

57

OX

R-3

69

AB

C-1

76

FO

X-3

29

CY

P-2

72

DU

OX

-31

5Z

BT

B-8

6S

GK

-40

6A

PA

F-3

52

XIA

P-3

18

PR

EX

-41

2

Cq

Target gene/amplicon length (bp)

Brilliant III

Company T

Brilliant III Ultra-Fast SYBR Green Delivers Improved

Reliability

37.13

31.80

NOTE: Reliability comparison of Brilliant III SYBR Green QPCR Master Mix and Company T master mix on 96 cDNA

targets on ABI 7500 Fast. Master mixes were run under recommended cycling conditions. Brilliant III total run time: 30

min. Company T total run time: 35 min. %GC for the 96 amplicons ranges from 30.4% to 69.5%

Brilliant III Ultra-Fast Performance Under Fast Cycling

Brilliant III Competitor B (Second fastest MM on the market)

Total cycling Time: 36.5 minutes Total cycling time: 37.5 minutes(1x 3min@95°C, 40x 5sec95°C/5sec@60°C) (1x 2min@98°C, 40x 5sec@98°C/5sec@60°C)

Eff. 92.9%; R2=0.997 Eff. 110.8%; R2=0.977

NOTE: NUMB target (305bp) was amplified from 10-fold dilutions of human gDNA at concentrations ranging from 50ng to

5pg/Rxn on BioRad CFX96.

Brilliant III vs. Competitor E’s Fast qPCR MM Across

Multiple TargetsGUS TBP

Comp E’s

Fast qPCR

Brilliant

III Ultra-

Fast

Cyclophilin

Eff.=87% Eff.=92% Eff.=83%

Eff.=94%Eff.=95%Eff.=97%

Brilliant III MM vs. Competitor R’s Fast MM Across Multiple

Targets

GUS TBP Cyclophilin

Comp

R’s Fast

Brilliant

III Ultra-

Fast

Eff.= 87% Eff.= 109% Eff.= 73%

Eff.= 97% Eff.= 95% Eff.= 94%

Brilliant III Ultra-Fast Generates Earlier Cts (Cqs) and Better

Reproducibility Across Varying Targets

GUS TBP CyclophilinCompetitor R Fast


Eff.= 86% Eff.= 96% Eff.= 82%

Eff.= 99% Eff.= 93% Eff.= 98%

Brilliant III Ultra-Fast Master Mix generates Ct values ~8 cycles earlier than the competitor for the Cyclophilin target – this

represents >2 orders of magnitude difference in detection.

brilliant qpcr reagents - agilent · brilliant iii ultra-fast products key product features 1. the...

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