perfect real time pcr products guide - ozyme · cell amptm direct rna prep kit for rt-pcr (real...

Perfect Real Time PCR Products GuideChoose the best Real Time PCR solution for your application !

TAKARA BIO EUROPEwww.takara-bio.eu

[email protected] • [email protected]: Europe: +33(0)1 3904 6880 • Austria: 0800 296 141 •

Germany: 0800 182 5178 • Switzerland: 0800 563 629 •UK: 0808 234 8063 • Fax: +33(0)1 3904 6870

• DNA & RNA template preparation

• One Step and Two Step Real Time RT-PCR

• Real Time PCR Premixes

• PrimerArrayTM Series

Table of content

Template preparation for RT-qPCR 2 Cell AmpTM Whole Transcriptome Amplification Kit (Real Time) (3730) 2 Cell AmpTM Direct RNA Prep Kit for RT-PCR (Real Time) (3732) 2 Cell AmpTM Direct Prep Kit for RT-PCR (Real Time) & Protein Analysis (3733) 3 EASY Dilution Solution (for qPCR) (9160) 3 For Dilution of DNA or RNA used in qPCR

One Step RT-qPCR Kits 4 One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) (RR066A) 4 One Step SYBR® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (RR086A) 4 One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (RR064A) 4

2-Step RT-qPCR Kit 5 PrimeScript™ RTase (2680A) 5 A new generation of Reverse Transcriptase

PrimeScript™ RT Reagent Kit (Perfect Real Time) (RR037A) 6 For cDNA Synthesis to be used as Template in qPCR

Real Time PCR Premixes 7 qPCR Premixes overview 7 SYBR® Premix Ex TaqTM (Perfect Real Time) (RR041A) 8 SYBR® Premix Ex TaqTM II (Perfect Real Time) (RR081A) 8 SYBR® Premix DimerEraserTM (Perfect Real Time) (RR091A) 8 SYBR® Premix Ex TaqTM (Tli RNase H Plus) (RR420A) 8 SYBR® Premix Ex TaqTM II (Tli RNase H Plus) (RR820A) 8

Premix Ex Taq™ (Perfect Real Time) (RR039A) 10 For qPCR using TaqMan® Probe Detection Method

Transgene Detection Primer Set for Real Time (Mouse) (3788) 10

Gene expression analysis 11 PrimerArrayTM Series 11 For pathways study (human & mouse)

Licences 14

Ordering and contact Informations 15

1

Template preparation for RT-qPCR

• Direct cDNA amplification, from small amount of cells.

• Efficient amplification of cDNA, without purifying RNA or cDNA.

• Good representativity : little bias included by amplification.

Cell AmpTM Whole Transcriptome Amplification Kit (Real Time) (3730)

This kit is designed to perform cDNA synthesis amplification directly from small amounts of cells. The amplified cDNA can be applied for real-time PCR as a template. In general, there is a problem in loss of nucleic acids during the purification process when dealing with small amounts cells. Moreover, cDNA can be amplified efficiently without purifying RNA or cDNA by using this kit. First, in this kit, cell lysis is performed, followed by synthesiz of cDNA from mRNA by reverse transcription using dT adapter primer (RT dT Primer). Next, add dA-tail by Terminal Deoxynucleotidyl Transferase (TdT) to synthesized cDNA, which will be used as a template for low cycle PCR to amplify the cDNA. This kit is useful to directly amplify cDNA from cells, and also to amplify cDNA from small amount of RNA. Takara’s Ex Taq™ HS (RR006A) is recommanded for efficient and consistent amplification of low and highly represented cDNA.

• Easy and fast preparation of template for 1- or 2- step real time RT-PCR: 10 minutes preparation without RNA extraction process from cell cultured.

• Efficient removal of genomic DNA.

• Compatible with high throughput gene expression analysis.

Cell AmpTM Direct RNA Prep Kit for RT-PCR (Real Time) (3732)

The product is a kit designed to prepare templates for real-time RT-PCR from animal cells cultured in 96-well plates or various types of plate with simple procedures, eliminating particular RNA extraction. The kit allows templates to be prepared from culture cells in just 10 minutes. In addition, a combination of the kit with 1-step real-time RT-PCR reagents such as One Step SYBR® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (RR086A/B) allows gene expression analysis to be completed in 2 hours or so.

CellAmp TM Add Processing Solution ＊→ Leave at RT for 5 minutes→ Collect dissolved cells

＊CellAmpTM Processing Bufferadded with DNase I　

Wash cells with Washing Buffer Incubate 5 min. at 75°C

Template ready for subsequent real-time RT-PCR !

Gene expression analysisusing real-time RT-PCR＊

＊RT-qPCR kit not supplied (seeordering information for appropriate kits)

Gene expression analysis completed in 2 - 2.5 hours only!

Flow chart Easy steps of template preparation

With a combination of the kit with a reverse transcription reagent, PrimeScript™ RT reagent Kit (Perfect Real Time) (RR037A/B), cDNA templates ready for real-time PCR can be synthesized in 30 minutes or so, significantly contributing to efficient high-throughput analysis. In addition, template samples prepared with CellAmp™ Direct RNA Prep Kit for RT-PCR (Real Time) are compatible with any of SYBR® Green assay and TaqMan® probe assay, and thus can serve a wide variety of experimental applications.

Target: Mouse Rplp1Reagent: SYBR® Premix Ex Taq™ II (Perfect Real Time)

Not available in the United States

Mouse cell

Total RNA from mouse liver

ApplicationAmplification of cDNA from mouse cell (2, 20, or 200 cells) and mouse total RNA (20 pg, 200 pg or 2 ng) were performed. The obtained cDNA amplified products were diluted to 1/10, and 2 μ l of those was used as PCR template. The figures show the result of Mouse Rplp1 which was obtained by performing real-time PCR. The template used for real-time PCR corresponds to mouse cell (8 × 10 -4, 8 × 10 -3, 8 × 10 -2 cells) and mouse total RNA (8 × 10 -3 pg, 8 × 10 -2 pg, 8 × 10 -1 pg) when re-calculated to initial template amount. This result shows that efficient amplification of cDNA was performed.

2

• Cell lysate directly used for both analysis of gene expression and protein expression.

• Easy and fast preparation of lysate for 1- or 2- step real time RT-PCR.

• No needs to purify nucleic acids or extract proteins.

Cell AmpTM Direct Prep Kit for RT-PCR (Real Time) & Protein Analysis (3733)

15URL:http://www.takara-bio.com

SampleReal Time RT-PCR

Undiluted Cell Lysate

Hmox1 westernblot analysis

：Reagent

EquipmentGene

：

：：

One Step SYBR ® PrimeScriptTM

Thermal Cycler Dice TM Real Time SystemMouse Hmox1Mouse Gapdh

Primer Designed by Perfect Real Time support system.

Standard Protocol

：

35Ct （

2nd

Der

ivat

ive

Max）

30

25

20

15

Before induction 1 2 4 6 8Time after induction (hours)

Western Blot

(Perfect Real time) RT-PCR Kit

Application RAW264.7 cells were stimulated by a Hmox1 inducing drug, and cell lysates prepared using this kit were used to analyze time-dependent changes in expression level of Hmox1 mRNA and protein.

Results /ConclusionAs shown in the results of real time RT-PCR, it was confirmed that the Ct values of Hmox1 decreased in time dependant manner after stimulation. Thus the expression level of Hmox1 mRNA increased. In addition, the increase of the expression level of Hmox1 protein was also confirmed by Western blotting. This result correlates with Hmox1 mRNA higher expression level in time depending manner after stimulation.

*Not available in US

Template preparation for RT-qPCR

This product is a kit to quickly prepare cell lysate from cultured cells that can be directly utilized for both analysis of gene expression by real-time RT-PCR, and protein expression by Western blot. Using simple protocol, cell lysate can be prepared within 10 minutes. The prepared cell lysate can be used as template in Takara Bio real time RT-PCR kits, and as sample for Western blots, without the need for additional steps to purify nucleic acids, or extract proteins. The included Loading Buffer does not require the addition of 2-mercaptoethanol to perform Western blot.

In addition, lysate prepared with this kit and used with an easy-to-use 1-step real-time RT-PCR kit, such as One Step SYBR® PrimeScript™ RT-PCR Kit II (Perfect Real Time) (RR086A/B)* allows completion of the whole analysis process in less than two hours. This kit can also be used to prepare template cDNA for real-time PCR in only 30 minutes when combined with Takara Bio’s reverse transcription kit such as PrimeScript™ RT reagent Kit (Perfect Real Time) (RR037A/B)*. The lysate prepared with this kit combines easily with many Takara Bio real time RT-PCR related products.

Takara’s EASY Dilution Solution is used to dilute template DNA or RNA when preparing serial dilutions of the standard for establishing the standard curve in real time PCR. When template DNA and RNA are diluted with sterilized water or TE buffer, they may stick to the inside of the tube which can prevent correct dilution. This product

helps to avoid this absorption and allows correct dilutions at lower concentrations. In addition, this product is RNA free, and will not contain non-specific amplification derived from RNA. Easy Dilution has been tested with Takara Bio’s real-time PCR reagents.

• Solution for qPCR Standard Dilution.

EASY Dilution Solution (for qPCR) (9160)For Dilution of DNA or RNA used in qPCR

3

One Stepts PrimeScript RT-PCR kits include: One of the following premixes: RR064A One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) for TaqMan® probe detection RR066A One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) RR086A One Step SYBR® PrimeScript™ RT-PCR Kit II (Perfect Real Time)

Content: - 2X optimized buffer for One-step RT-PCR (3x840 μL) including dNTP mix and Mg2+. SYBR® Green I is also included in buffers of RR066A and RR086A. - TaKaRa Ex Taq™ HS DNA Polymerase (100 μL ; 5U/ μL) or included in the Enzyme Mix of RR086A. - PrimeScript™ RTase and RNase inhibitor included in an Enzyme Mix (100 μL in case of RR064A or RR066A, 200 μL for RR086A whith Ex Taq HS) - RNase free dH2O (1.25 mL x 2) - All contain separate tube of ROX™ Reference Dye I (50× conc.; 100 μL) and ROX™ Reference Dye II (50× conc.; 100 μL).

Standard curve (amplification in background) Melting curve

Performance of Real-Time 1 step RT-PCR with SYBR® Green I detection


• Efficient: Uses an RTase with strong elongation activity and high performance Ex Taq™ polymerase.

• High Specificity: Improved buffer and hot start polymerase.

• Highly Sensitive: Accurate quantification of low abundance RNA.

• Easy protocol: One step RT-qPCR lowers pipeting and contami-nation risks.

• Ideal for gene expression studies

One Step RT-qPCR Kits

These products are used for one-step qRT-PCR using PrimeScript™ RTase as RT and Takara’s Ex Taq™ HS for qPCR. The 2 first products are for SYBR® Green I detection and the later is for TaqMan® probe detection. These products have excellent amplification rates and reaction specificities and are best used for RNA viruses or other small RNA detections. One Step SYBR® PrimeScriptTM RT-PCR Kit II has an

improved system buffer which results increasing reaction specificity. Thus by reducing non-specific and primer-dimer amplification most accurate quantification is obtained. In addition the kit achieves a simple and convinient procedure by using premixed components.

Application Detection of Rat Rplp2 (ribosomal protein, large, P2) mRNA from total RNA using One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time). Template: Rat liver total RNA 6.4 pg - 100 ng (negative control is dH2O)Primers: Specific primers supplied through custom service (Perfect

Real Time Support System, only available in Japan)

RT and PCR reaction conditions 1) RT step: 42°C 5 min. 95°C 10 sec. 2) PCR step: 40 cycles: 95°C 5 sec. 60°C 30 sec. 3) Melting step: 60°C to 94°C Thermal Cycler Dice™ Real Time System (TP800)* was

used as Real Time PCR instrument

* TP800 is not available in the U.S. or Europe

Results /ConclusionTarget gene can be detected in the range of 6.4 pg-100 ng total RNA. Good linearity of the standard curve was obtained. This indicates that One Step SYBR® PrimeScript™ RT-PCR Kit (Perfect Real Time) achieved accurate quantification in the whole range of RNA concentrations. The melting curve has a single peak demonstrating excellent specificity for this kit.

Limited Use Label License: RR066A, RR086A : [P5] [L1] [L11] [L15] [L46] [M57]RR064A: [P7] [L1] [L15] [M57]

4

One Step PrimeScript™ RT-PCR Kits For simple RNA Quantification in One Step

2-Step RT-qPCR Kits

Pre-incubate at

42°C or 50°C

then add RTase

Add

RTase on ice

65°C

0°C

42°Cor

50°C

Denature RNA

and add

RTase on ice

Protocol

Oligo dT primer

dNTP

Total RNA

(0.1; 1; 10 ng)

Add RTase PrimeScriptTM

(RT at 42°C)

SuperScr ip t ® I I I

(RT at 50°C)

PCR

RT (15-30 minutes)RTase inactivation

Target:

Dystrophin 8 kb

A New Generation of Reverse Transcriptase

• Strong Displacement Extension Activity, makes it capable of extending longer templates.

• cDNA Synthesis with Minimal Background & high quality product at 42°C

• High Accuracy Reverse Transcription

PrimeScript™ RTase (2680A)

Comparison of PrimeScript vs SuperScript III on a Long, Highly Structured RNA. The addition of RTase after the RNA is denatured is used to avoid suppression of cDNA synthesis. PrimeScript™ shows the ability to amplify cDNA at every step of this process even when on ice.


PrimeScript™ RTase is a RTase from Takara Bio which was originally developed from M-MLV RTase. This enzyme possesses excellent extension ability and synthesizes 1st strand cDNA with high sensitivity and yield. In addition, it can reverse transcribe complex, highly structured RNA at the normal reaction temperature of 42°C. Non-specific annealing of RTase to RNA can interfer with cDNA synthesis during reverse transcription, particularly with highly structured RNA. The resulting non-specific product can have a negative effect on RT efficiency or full length cDNA synthesis. To minimize the above factors, a high temperature reaction is often used

to relax the secondary structures. However, the high temperature may cause RNA degradation which is not ideal. PrimeScript™ RTase is an enzyme that reduces inefficient cDNA synthesis and mispriming by controlling non-specific annealing and optimizing RTase priming to double-stranded portions of template RNA and RT-primer. Furthermore, it contains strong displacement activity. So, this enzyme is capable of synthesizing cDNA with minimal background for complex structures of RNA or longer targets of RNA at 42°C, and is less damaging than other RTase’s.

5

Application A comparison of 2-step real time RT-PCR (qRT-PCR) with TaqMan® Probe detection by using the PrimeScript™ RT reagent kit (Perfect Real Time) or ABI’s Reagent for 1st Strand cDNA Synthesis was performed. For the RT reaction, serially diluted Human liver total RNA (500 fg to 500 ng) was used in 10 µl reactions using random hexamers and oligo dT primers. RT conditions used with each kit are described in the figure. TaqMan® Probe Detection. Premix Ex Taq™ (Perfect Real Time) (RR039A) was used for amplification in a Thermal Cycler Dice Real Time System ( TP800)*.

TaqMan® Probe DetectionPrimeScript™ RT Reagent Kit (Perfect Real Time) ABI Reagent for 1st Strand cDNA Synthesis

Results /ConclusionGood linearity and excellent efficiency was obtained in 2-step qRT-PCR using both the PrimeScript™ RT reagent kit (RR037A) and ABI’s Reagent. However, Takara’s PrimeScript™ RT reagent’s reaction was completed in approximately 15 minutes, which is 1/4 of the time of the ABI reagent reaction. PrimeScript™ RT Reagent Kit provides excellent efficiency and fast reaction times for superior real time PCR reactions.

• Fast and Efficient: Complete RT reaction in 15 minutes.

• Sensitive: Suitable for two-step qRT-PCR.

• Convenient: Contains random and oligo dT primers.

PrimeScript™ RT Reagent Kit (Perfect Real Time) (RR037A)For cDNA Synthesis to be used as Template in qPCR

2-Step RT-qPCR Kit

RT conditions

37°C 15 min. 85°C 5 sec.

(Approx. 15 min.)

RT conditions

25°C 10 min. 37°C 60 min. 85°C 5 sec. ;

according to manufacturers recommendations

(Approx. 70 min.)

*TP800 is not available in US or Europe

Template: 2 µL of RT reaction Target: Human GAPDH Reaction Volume: 25 µLPrimer/Probe: TaqMan® Gene Expression Assay (VIC labelled) (Applied Biosystems)PCR Conditions: Initial denaturation 95°C 10 sec. Then: 40 cycles: 95°C 5 sec. and 60°C 30 sec.


The PrimeScript™ RT Reagent Kit (Perfect Real Time) is a 1st strand cDNA synthesis kit for qPCR featuring PrimeScript™ RTase, for robust reverse transcription on any RNA template. The kit is simple to use and includes universal primers and all necessary reagents. Sufficient

cDNA for qPCR is obtained in a 15 minutes reaction. This kit is suitable for high throughput analysis in combination use with SYBR® Premix Ex Taq™ (Perfect Real Time) series for SYBR® Green detection or Premix Ex Taq™ (Perfect Real Time) for TaqMan® probe detection.

6

qPCR Premixes overview

7

qPCR Product Selection

Takara’s qPCR enzyme premixes encompass the performance characteristics required for qPCR. These enzymes will provide comparable high performance when using many standard templates and primers; however, there are situations in which one enzyme will outperform the others. Please review the table below to help with your qPCR enzyme premix selection.

Fast High-Sensi-tivity

High-Speci-ficity

Ready-to-use Premix

Use on any qPCR instru-ment

Limit Non-Specific Amplification

Limit Primer Dimers

Minimize Inhibition from Residual mRNA

Premix Ex Taq™ (Perfect Real Time) (Cat. # RR039A)

XXX XXX XXX XXX XXX --- --- ---

SYBR® Premix Ex Taq™ (Perfect Real Time) (Cat. # RR041A)

XXX XXX XX XXX XXX --- --- ---

SYBR® Premix Ex Taq™ II (Perfect Real Time) (Cat. # RR081A)

XXX XXX XXX XXX XX XXX XXX ---

SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Cat. #RR420A)

XXX XXX XX XXX XXX --- --- XXX

SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat. # RR820A)

XXX XXX XXX XXX XX XXX XXX XXX

SYBR® Premix Dimer- Eraser™ (Perfect Real Time) (Cat. # RR091A)

X XXX XXX XXX XX XXX XXX ---

• SYBR® Premix Ex Taq™ II (Perfect Real Time) (Cat. # RR081A) and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat. # RR820A) should be se-lected when using primers that tend to form dimers or display non-specific binding

• SYBR® Premix Ex Taq™ (Perfect Real Time) (Cat. # RR041A) and SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Cat. # RR420A) have faster extension rate than respective SYBR® Premix Ex Taq™ II. They are recommended when target is longer than 400 bp.

• SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat. # RR820A) offers more specificity than SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Cat. # RR420A) and should be selected when amplifying a GC-rich template.

• SYBR® Premix DimerEraser™ (Cat. # RR091A) offers highest specificity, however 3 step PCR is required. It should be used whenever non specific amplification is detected and it is not possible to change primers

Probe DetectionSYBR Detection

1. Reverse Transcription for qPCR

2. qPCR

• Select from various premixes for most accurate quantification from different template types and quantities: See selection table.

• Fast Reaction Time: Shorter reaction times due to optimized buffer. Applicable for use with Fast PCR instruments

• High Sensitivity: Detects low copies of target.

• Wide Dynamic Range.

• Accurate Quantification: Produces excellent standard curves using numerous real time instruments.

• Convenient: Separate tubes of ROX reference dye are supplied for compatibility with most instruments on the market.

Amplification curve Melting curve

Results /ConclusionReaction specificity was compared between the SYBR® Premix Ex Taq™ II and competitor’s products using same pair of primers. Each PCR was performed according to the standard protocol of each product. Results show that SYBR® Premix Ex Taq™ II achived complete suppression of non-specific amplifications and so the specificity is increased.

Limited Use Label License : [P5] [L11] [L15] [L46] [M57]

Figure 1Target: Human TFRCTemplate: Serial dillution of cDNA (equivalent to HL60 cells total RNA 10 pg - 100 ng; 1 reaction each) or H2O (NTC, in duplicate) Instrument: Thermal Cycler Dice™ Real Time System

SYBR® Premix Ex TaqTM II (Perfect Real time)PCR conditions: Optimal 2- step protocol95°C 30 sec. } 1 cycle

95°C 5 sec. 60°C 30 sec. 40 cycles

FastStart Universal SYBR® Green MasterPCR conditions:95°C 10 min. } 1 cycle

95°C 10 sec 60°C 30 sec 40 cycles

Real Time PCR Premixes

Using high quality reagents for qPCR minimizes optimization and improves assay consistency. SYBR® Premix Ex Taq™ products are unsurpassed for high-speed, high-specificity application results. These 2× concentration premixes combine the high performance of Takara’s Ex Taq™ Hot-Start DNA Polymerase, with an optimized real time PCR buffer including SYBR® Green I, which provides superior specificity and increased amplification efficiency. Separate tubes of ROX reference dye are also included. Excellent qPCR results are obtained quickly and easily. Excellent real time PCR results are obtained quickly and easily.Both SYBR® Premix Ex Taq™ (Perfect Real Time) result in quick reaction according to the 2 steps standard protocol. First is used for great efficiency and in cases where real time PCR is difficult. On the other hand, version II is used when higher specificity is required by suppressing non-specific amplification such as primer-dimer.

Concerning SYBR® Premix DimerEraser™ , it has an improved buffer system compared to both previous premixes, which is added with the original accessory protein and results in further suppression of non-specific amplification.The two new SYBR premixes for Real-Time PCR, including the thermostable Tli RNAse H are based on the existing highly efficient SYBR® Premix Ex Taq™ (Perfect Real Time). They were developed to address the inhibitory effect on the PCR due to the presence of remaining RNA after cDNA synthesis. This inhibition is seen particularly for GC rich templates and genes with poor expression.Tli RNase H added to the qPCR premix removes the inhibition, without compromising the efficiency of the reaction, as shown in figures 2 and 3. Therefore these new premixes allow excellent quantification of any type of cDNA and thus unparalleled accuracy in gene expression studies.

RR081A

Roche Premix

ABI Premix

Non Speci�c ampli�cation

Non Speci�c ampli�cation

SYBR® Premix Ex TaqTM Products For qPCR using SYBR® Green I Detection

Fast SYBR® Green MasterPCR conditions:95°C 20 sec. } 1 cycle

94°C 10 sec 60°C 20 sec 40 cycles

8

SYBR® Premix Ex Taq™ products include: One of the following premixes: RR041A SYBR® Premix Ex Taq™ (Perfect Real Time) (2× conc.)* 5 × 1 mL RR081A SYBR® Premix Ex Taq™ II (Perfect Real Time) (2× conc.)* 5 × 1 mL RR091A SYBR® DimerEraser™ (Perfect Real Time) (2× conc.)* 5 × 1 mL RR420A SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (2× conc.)** 5 × 1 mL RR820A SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (2× conc.)** 5 × 1 mL

*Contains TaKaRa Ex Taq™ HS DNA Polymerase, dNTP mix, Mg2+ and SYBR® Green I. **SYBR® Premix Ex Taq™ (Tli RNaseH Plus) and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) also contain Tli RnaseH. SYBR® Premix Ex Taq™ II and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) contain an optimized buffer to limit primer dimer formation and non-specific amplification. All contain separate tube of ROX™ Reference Dye I (50× conc.; 200 μL) and ROX™ Reference Dye II (50× conc.; 200 μL).

9

Target： C16orf84 (GC 73%)

◆ RR420

■A(1)

primer dimer

◆ RR420

▲Bout of range

Target： ATP5F1 (GC 45%)

out of standard curve

◆ RR420 ■company A(1) ▲company B

Target： PGK1 (GC 54%)

◆ RR820 ■company A(2) ▲company C

Target： PLSCR3 (GC 67%)

E�.＝9.4％E�.＝77％E�.＝88％

E�.＝94％E�.＝77％E�.＝88％

Template：Human testis cDNA（corresponding to total RNA 10 pg ～ 100 ng）


RR820

A(2)

C

30 sec.(2 step)

60 sec.(2 step)

30 sec.(3 step)

1 h 25 min.

2 h 1 min.

2 h 13 min.

elongation time reaction time

40

35

30

25

CtCt

20

15

20

22

24

26

28

30

32

34

36

38

40

1.E＋00 1.E＋02

template RNA（pg）


1.E＋04

1.E＋00 1.E＋02 1.E＋04

Ct

20

22

24

26

28

30

32

34

36

38

40


1.E＋00 1.E＋02 1.E＋04

Ct

15

20

25

30

35

40


1.E＋00 1.E＋02 1.E＋04

unspecific


Target genes：

A : LSP1 (GC 68%)

B : C16orf84 (GC 73%)

Figure 2Comparison of Tli RNase H added premix (RR420) with its existing counterpart without RNase H (RR041) . With the new products, RR420 & RR820, no inhibition of PCR by the remaining mRNAs can be detected with higher concentrated cDNA templates and thus precise quantification can be possible with much broader dynamic range. This can be observed more significantly in case of low expressed genes of high GC content. This is due to the activity of the thermo-stable Tli RNase, premixed with the reaction solution, which helps degrade remaining mRNAs in the template cDNA solution.

Figure 3Comparison of RR420 and RR820 versus competitors premixes. Tli Plus premixes allow better quantification over a broader dynamic range of concentrations. In addition RR820 shows more efficient amplification with faster reaction speed.


• Instrument Compatibility: Compatible with most real time PCR instruments on the market.

• High Sensitivity: Detects low copies of target.

• Wide Dynamic Range.

• Accurate Quantitation: Produces excellent standard curves with numerous real time instruments.

• Convenient: Separate tubes of ROX reference dye are supplied.Amplification Curve

Standard Curve

Amplification Curve

Standard Curve

Results /ConclusionTakara’s Premix Ex Taq™ (Perfect Real Time) requires half the time of the TaqMan® Universal PCR Master Mix with the TaqMan® Gene Expression Assays to achieve excellent results for this real time PCR application.

Limited Use Label License : [P7] [L15] [M57]

Premix Ex Taq™ (Perfect Real Time) is a 2X premix, specially designed for high speed, high sensitivity real time PCR using TaqMan® probes. This premix includes Takara’s high-performance Ex Taq™ hot Start DNA Polymerase, which uses antibody-mediated Hot Start technology to prevent non-specific amplification. In addition, an optimized real time PCR buffer provides increased amplification efficiency and further improved specificity for high speed real time PCR. Excellent PCR real time results are obtained quickly and easily.

Takara Premix Ex Taq™ (Perfect Real Time)PCR conditions:95°C 10 sec. } 1 cycle

95°C 5 sec. 60°C 34 sec.

(Approx. 50 min.)

40 cycles

Takara Premix Ex Taq™ (Perfect Real Time) Applied Biosystems TaqMan®

Universal PCR Master Mix

ABI’s TaqMan® Universal PCR Master MixPCR conditions:95°C 10 min. } 1 cycle

95°C 15 sec. 60°C 1 min.

(Approx. 90 min.)

40 cycles

Premix Ex Taq™ (Perfect Real Time)(RR039A)For qPCR using TaqMan® Probe Detection Method


Application an® Gene Expression Assays (Applied Biosystems) Template: cDNA seriaTarget: Human ACTB Probe/Primer: TaqMl dilution (corresponding to total RNA 1 pg - 100 ng)

Transgene Detection Primer Set for Real Time (Mouse) (3788)

• Detection and quantification of transgene inserted in transgenic mouse genome.

This kit is a primer set for real-time PCR to detect GFP (EGFP, AcGFP1) and lacZ, which are widely used as gene marker inserted in transgenic mice. It is possible to screen transgenic mice by detecting these marker genes with real-time PCR. The primer sets (Ywhaz, Raver2) for detecting genes on mouse genomic DNA are also included as a reference.

Therefore, it is possible to make a comparison of the transgene contents between samples by using the relative quantification method.Takara Bio recommends the use of SYBR® Premix Ex Taq™ (Perfect Real Time) (RR041A/B) and SYBR® Premix Ex Taq™ II (Perfect Real Time) (RR081A/B) with this primer set.

10

PrimerArrayTM Series

Gene expression analysis

The PrimerArray™ is a set of real-time RT-PCR primers for the analysis of gene expression associated with specific biological pathways. Each array contains a mixture of 96 primer pairs for 88 pathway related genes and 8 housekeeping genes. When quantifying an unknown sample against a control sample, genes will show differential expression via the relative quantification method, and multiple

expression levels can be screened simultaneously. The PrimerArray™ Analysis Tool Ver. 2.0 is useful for analysis with this product. The tool performs relative quantification analysis by ΔΔCt method using Ct values calculated by the software of a real-time PCR instrument, and displays the graphical results.

• Economical: Enough reagents for 12 reactions/well.

• One shot screening of 88 varieties of pathway related genes: Using the difference in gene expression level.

• Pre-optimized for superior performance with SYBR® Premix Ex TaqTM

• Easy Analysis: With a tool exclusive for use with PrimerArrayTM products.

Steps for Preparation1). RNA extraction (approx. 30 min): Extract RNA from experimental materials (control sample and test sample) using the FastPure® RNA

Kit ( 9190), and treat with DNase I. Use approx. 2.5 μg of total RNA for one experiment.

2). Reverse transcription (approx. 20 min): Synthesize cDNA from each of the total RNA samples. Takara recommends use of the Blue-Print™ RT reagent Kit (for real time)( RR737A) or PrimeScript (RR037A).

3). Dispense the real-time PCR reaction solution (approx. 10 min): Combine the synthesized cDNA and SYBR® Premix Ex Taq™ II (Perfect Real Time) to prepare a master mix solution for control and test samples, then dispense into wells of a 96-well real-time PCR plate. (see illustration below)

For Control Sample

Master mix

For Test Sample

Master mix

PrimerArray ™

For Test Sample

For Control Sample

4). Add primers (approx. 5 min): Add primers from the PrimerArray™ plate to the real-time PCR plate using a multichannel pipette, etc.

How to use the PrimerArray™ Series A flowchart starting with RNA extraction ending with data analysis is laid out in the following diagrams. The time in parentheses is an estimate of time required for one real-time PCR experiment,

and the entire process can be finished in approximately 2.5 to 3.5 hours. Multiple samples can be processed together to the reverse transcription reaction step, and the synthesized cDNA should be stored at -20°C for subsequent experiments.

11

Control Sample Test Sample

5). Perform Real-time PCR (1-2 hours): The reaction is carried out with a real-time PCR instrument.

6). Data analysis is done using the PrimerArray™ Analysis Tool (about 30 min): Relative quantification analysis is performed using the ΔΔ Ct method.

The PrimerArray™ Analysis Tool Ver. 2.0 is a software tool for analysis of data obtained using Takara’s PrimerArray™ series ( PH001-PH010, PN001-PN010), allowing comparison of an unknown and control sample. It performs relative quantitative analysis using Ct values calculated using the real time PCR instrument software by the ΔΔCt method, and the result is supplied in a graphical format. Further analysis can be easily performed using the KEGG pathway analysis tool (see page 4).

• The PrimerArray™ Analysis Tool Ver. 2.0 uses an Excel® format file that contains macros. Its performance has been validated in the following OS (operating systems) and versions of Microsoft® Office Excel®:

Windows® XP operating systemMicrosoft® Office Excel® 2003Microsoft® Office Excel® 2007

• The PrimerArray™ Analysis Tool Ver. 2.0 is available for download from the ara Bio Website.

http://catalog.takara-bio.co.jp/en/IMAGES/primerarray_tool_v2_en.zip

Details on the analysis software can be found in the PrimerArray™ Analysis Tool Ver. 2.0 manual. Samples of Scatter Plots and 3D profiles are shown below and include the information about using the KEGG pathway which is a simple visual reference for seeing changes in gene expression related to the pathway of interest. http://catalog.takara-bio.co.jp/en/PDFS/primerarray_analysis_tool-v2_en.pdf

Scatter PlotThe figure to the right shows an example of a scatter plot using the PrimerArray™ Analysis tool. The left table shows a list of quantitated values and standard deviation before relative quantitation against the control sample. Those values are then shown in the scatter plot in the graph to the right.

Scatter Plot 3D Profile


PrimerArrayTM Analysis Tool and How to use it

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Link to KEGG pathway (from the PrimerArrayTm analysis software) View of KEGG pathway

What is KEGG ?KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database of biological systems, consisting of genetic building blocks of genes and proteins (KEGG GENES), chemical building blocks of both endogenous and exogenous substances (KEGG LIGAND), molecular wiring diagrams of interaction and reaction networks (KEGG PATHWAY), and hierarchies and relationships of various biological objects (KEGG BRITE). Since 1995 KEGG has been

developed by the Kanehisa Laboratories in the Kyoto University Bioinformatics Center and the Human Genome Center of the University of Tokyo as part of their research activities. KEGG provides a reference knowledge base for linking genomes to biological systems and also to environments by the processes of PATHWAY mapping and BRITE mapping.

See the website at http://www.genome.jp/kegg/

Visualize the Expression Level Changes using the KEGG Pathway

3D Profile (Fold Difference)The figure to the right shows the fold difference (the expression level of the unknown sample based on the expression level of the control sample equal to one) in the bar graph. Above the graph is a table listing the fold difference of the test sample and gene symbols, with the placement of the data corresponding to their positions on the plate.

The button for the KEGG pathway is included in the PrimerArray™ Analysis software as ara is an authorized service provider for this software. It will lead to further analysis of the experiment. See below for an explanation of the KEGG pathway.

Initially, a color-coded legend based on the difference of expression on the KEGG pathway map will be shown (as below). Clicking on the KEGG pathway button leads you to the next screen. (lower right corner).

Pathway map displaying the relative expression levels of the genes. In addition, the up and down regulated genes can be confirmed with this map.

1

2

Push this button to lead you to KEGG pathway


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1

2

Trademarks and Licensing [P1] : Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights (such as 5’ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [P5] PCR Notice: Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

[P7] PCR Notice: A license to perform the patented 5’ Nuclease Process for research is obtained by the purchase of (i) both Authorized 5’ Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5’ Nuclease Kit, or (iii) license rights from Applied Biosystems. This product is an Authorized 5’ Nuclease Core Kit. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,210,015, 5,487,972, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. Separate purchase of a Licensed Probe would convey rights under the applicable claims of US Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569, 5,804,375 (claims 1-12 only), and 6,214,979, and corresponding claims outside the United States. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

[L11] SYBR® Green I:This product is covered by the claims of U.S. Patent No. 5,436,134 and 5,658,751 and their foreign counterpart patent claims.Takara PCR products containing SYBR® Green I are sold under license from Molecular Probes Inc. only for the usage in Real-time PCR for internal research purpose. These products are not to be used for the purpose such as; providing medical, diagnostic, or any other testing, analysis or screening services or providing clinical information or clinical analysis in return for compensations.

[L15] Hot Start PCR: Licensed under U.S. Patent No. 5.338,671 and 5,587,287, and corresponding patents in other countries.

[L46] SYBR/Melting Curve Analysis: The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein.

[M57] LA Technology: This product is covered by the claims 6-16 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims.

[L1] One Step RNA PCR / One Step RT-PCR: Use of this product is licensed from bioMerieux, is covered by US Patent 5,817,465 and equivalents, and is for Research Use Only.

SYBR® is a trademark of Molecular Probes Inc. TaqMan® is a trademark of Roche Molecular Systems Inc. Registered names, trademarks, etc. used in this document are the property of their respective owners, even when not specifically marked as such.

NOTE: All products are intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC.

Licences

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Ordering Information (please see our website for the different kits’ components) Real Time PCR/RT-PCR products

Product name Product No. Quantity

SYBR® Premix Ex Taq™ RR041A 200 rxns (5 x 1 ml) (Perfect Real Time) RR041L* 200 rxns (5 ml)

RR041W* 1000 reactions (25 ml)

SYBR® Premix Ex Taq™ II RR081A 200 rxns (5 x 1 ml)

(Perfect Real Time) RR081L* 200 rxns (5 ml)

RR081W* 1000 rxns (25 ml

SYBR® Premix Ex Taq™ (Tli RNase H Plus) RR420A 200 rxns (5 x 1 ml)

SYBR® Premix Ex Taq™ II (Tli RNase H Plus) RR820A 200 rxns (5 x 1 ml)

SYBR® Premix DimerEraser™ (Perfect Real Time) RR091A 200 rxns (5 x 1 ml)

Premix Ex Taq™ (Perfect Real Time) RR039A 200 rxns (5 x 1 ml) RR039W 1000 rxns (25 ml)

Transgene Detection Primer Set 3788 100 rxns

for Real Time (Mouse)

EASY Dilution Solution 9160 8 x 1 mL

* Larger sizes (with references LR and WR) already added with ROX reference Dye are also available

ZZWTBE1103EU


PrimeScript™ Reverse Transcriptase 2680A 10,000 U

PrimeScript™ RT Reagent Kit (Perfect Real Time) RR037A 200 rxns

One Step PrimeScript™ RT-PCR RR064A 100 rxns

Kit (Perfect Real Time)

One Step SYBR® PrimeScript™ RR066A 100 rxns

RT-PCR Kit (Perfect Real Time)

One Step SYBR® PrimeScript™ RR086A 100 rxns

RT-PCR Kit II (Perfect Real Time)

Cell Amp™ Whole Transcriptome 3730 50 rxns

Amplification Kit (Real Time)

Cell Amp™ Direct RNA Prep 3732 200 rxns

Kit for RT-PCR (Real Time)

Cell Amp™ Direct Kit for RT-PCR 3733 200 rxns

(Real Time) & Protein Analysis

PrimerArrayTM Series products

Product name (Human) Product No. Quantity

PrimerArrayTM Cytokine-cytokine PH001 1 set*

receptor interaction

PrimerArrayTM Cell Cycle PH002 1 set*

PrimerArrayTM Cell adhesion molecules PH003 1 set*

PrimerArrayTM Jak-STAT signaling pathway PH004 1 set*

PrimerArrayTM Natural Killer cell PH005 1 set*

mediated cytotoxicity

PrimerArrayTM Axon guidance PH006 1 set*

PrimerArrayTM Focal adhesion PH007 1 set*

PrimerArrayTM T cell receptor PH008 1 set*

signaling pathway

PrimerArrayTM TGF-beta PH009 1 set*

signaling pathway

PrimerArrayTM Wnt signaling pathway PH010 1 set*

* enough reagent for 12 plates

Product name (Mouse) Product No. Quantity

PrimerArrayTM Cytokine-cytokine PN001 1 set*

receptor interaction

PrimerArrayTM Cell Cycle PN002 1 set*

PrimerArrayTM Cell adhesion molecules PN003 1 set*

PrimerArrayTM Jak-STAT signaling pathway PN004 1 set*

PrimerArrayTM Natural Killer cell PN005 1 set*

mediated cytotoxicity

PrimerArrayTM Axon guidance PN006 1 set*

PrimerArrayTM Focal adhesion PN007 1 set*

PrimerArrayTM T cell receptor PN008 1 set*

signaling pathway

PrimerArrayTM TGF-beta PN009 1 set*

signaling pathway

PrimerArrayTM Wnt signaling pathway PN010 1 set*

Recommended qPCR related products from Clontech


qPCR Human Reference Total RNA 636690 25 ug

qPCR Human Reference cDNA, oligo dT 636692 25 rxns

qPCR Human Reference cDNA, oligo dT 636693 100 rxns

qPCR Human Reference cDNA, random primed 639653 25 rxns

qPCR Human Reference cDNA, random primed 639654 100 rxns

New from Clontech – qPCR direct from tissue or dirty samples


Terra™ qPCR Direct SYBR® Premix 638318 400 rxnsTerra™ qPCR Direct SYBR® Premix 638319 200 rxns

For GC rich template / gDNA /epigenetic

SYBR® Advantage® GC qPCR Premix 638320 200 rxns

Contact Information:

Takara Bio EuropeWebsite: www.takara-bio.euE-mail: [email protected]: +33.(0)1 3904 6880Fax: +33.(0)1 3904 6870

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