Perfect Real Time PCR Products Guide

Transcription

1 Perfect Real Time PCR Products Guide Choose the best Real Time PCR solution for your application! DNA & RNA template preparation One Step and Two Step Real Time RT-PCR Real Time PCR Premixes PrimerArray TM Series TAKARA BIO EUROPE Tel: Europe: +33(0) Austria: Germany: Switzerland: UK: Fax: +33(0)

2 Table of content Template preparation for RT-qPCR 2 Cell Amp TM Whole Transcriptome Amplification Kit (Real Time) (3730) 2 Cell Amp TM Direct RNA Prep Kit for RT-PCR (Real Time) (3732) 2 Cell Amp TM 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 Taq TM (Perfect Real Time) (RR041A) 8 SYBR Premix Ex Taq TM II (Perfect Real Time) (RR081A) 8 SYBR Premix DimerEraser TM (Perfect Real Time) (RR091A) 8 SYBR Premix Ex Taq TM (Tli RNase H Plus) (RR420A) 8 SYBR Premix Ex Taq TM 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 PrimerArray TM Series 11 For pathways study (human & mouse) Licences 14 Ordering and contact Informations 15 1

3 Template preparation for RT-qPCR Cell Amp TM Whole Transcriptome Amplification Kit (Real Time) (3730) Not available in the United States Direct cdna amplification, from small amount of cells. Efficient amplification of cdna, without purifying RNA or cdna. Good representativity : little bias included by amplification. 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. Target: Mouse Rplp1 Reagent: SYBR Premix Ex Taq II (Perfect Real Time) Application Amplification 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, , cells) and mouse total RNA ( pg, pg, pg) when re-calculated to initial template amount. This result shows that efficient amplification of cdna was performed. Mouse cell Total RNA from mouse liver Cell Amp TM Direct RNA Prep Kit for RT-PCR (Real Time) (3732) 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. 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. 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. Add Processing Solution Wash cells withcellamp TM Washing Buffer Leave at RT for 5 minutes Collect dissolved cells Incubate 5 min. at 75 C CellAmp TM Processing Buffer added with DNase I Template ready for subsequent real-time RT-PCR! Gene expression analysis using real-time RT-PCR RT-qPCR kit not supplied (see ordering information for appropriate kits) Gene expression analysis completed in hours only! Easy steps of template preparation 2

4 Template preparation for RT-qPCR Cell Amp TM Direct Prep Kit for RT-PCR (Real Time) & Protein Analysis (3733) 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. 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-touse 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. *Not available in US 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 /Conclusion As 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. Western Blot Ct 2nd Derivative Max Hmox Standard Protocol Time after induction (hours) Real Time RT-PCR Sample Undiluted Cell Lysate Reagent One Step SYBR PrimeScript TM RT-PCR Kit (Perfect Real time) Equipment Thermal Cycler Dice TM Real Time System Gene Mouse Hmox1 Mouse Gapdh Primer Designed by Perfect Real Time support system. EASY Dilution Solution (for qpcr) (9160) For Dilution of DNA or RNA used in qpcr Solution for qpcr Standard Dilution. 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. 3

5 One Step RT-qPCR Kits One Step PrimeScript RT-PCR Kits For simple RNA Quantification in One Step 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 contamination risks. Ideal for gene expression studies 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 PrimeScript TM RT-PCR Kit II has an One Stepts PrimeScript RT-PCR kits include: Not available in the United States 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 Mg 2+. 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 dh 2 O (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). 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 ng (negative control is dh 2 O) 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 Standard curve (amplification in background) Melting curve Results /Conclusion Target 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. Performance of Real-Time 1 step RT-PCR with SYBR Green I detection Limited Use Label License: RR066A, RR086A : [P5] [L1] [L11] [L15] [L46] [M57] RR064A: [P7] [L1] [L15] [M57] 4

6 2-Step RT-qPCR Kits PrimeScript RTase (2680A) A New Generation of Reverse Transcriptase Not available in the United States 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 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. Protocol Oligo dt primer dntp Total RNA (0.1; 1; 10 ng) 65 C 42 C or 50 C 0 C Add RTase RT (15-30 minutes) RTase inactivation Denature RNA and add RTase on ice PrimeScript TM (RT at 42 C) Pre-incubate at 42 C or 50 C then add RTase Add RTase on ice SuperScript III (RT at 50 C) PCR Target: Dystrophin 8 kb 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. 5

7 2-Step RT-qPCR Kit PrimeScript RT Reagent Kit (Perfect Real Time) (RR037A) For cdna Synthesis to be used as Template in qpcr Fast and Efficient: Complete RT reaction in 15 minutes. Not available in the United States Sensitive: Suitable for two-step qrt-pcr. Convenient: Contains random and oligo dt primers. 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. 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 Detection PrimeScript RT Reagent Kit (Perfect Real Time) ABI Reagent for 1st Strand cdna Synthesis *TP800 is not available in US or Europe Results /Conclusion Good 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. 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.) Template: 2 µl of RT reaction Target: Human GAPDH Reaction Volume: 25 µl Primer/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. 6

8 qpcr Premixes overview 1. Reverse Transcription for qpcr 2. qpcr SYBR Detection Probe Detection 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. Premix Ex Taq (Perfect Real Time) (Cat. # RR039A) SYBR Premix Ex Taq (Perfect Real Time) (Cat. # RR041A) SYBR Premix Ex Taq II (Perfect Real Time) (Cat. # RR081A) SYBR Premix Ex Taq (Tli RNaseH Plus) (Cat. #RR420A) SYBR Premix Ex Taq II (Tli RNaseH Plus) (Cat. # RR820A) SYBR Premix Dimer- Eraser (Perfect Real Time) (Cat. # RR091A) Fast High-Sensitivity High-Specificity Ready-to-use Premix Use on any qpcr instrument Limit Non- Specific Amplification Limit Primer Dimers Minimize Inhibition from Residual mrna XXX XXX XXX XXX XXX XXX XXX XX XXX XXX XXX XXX XXX XXX XX XXX XXX --- XXX XXX XX XXX XXX XXX XXX XXX XXX XXX XX XXX XXX XXX 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 selected 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 7

9 Real Time PCR Premixes SYBR Premix Ex Taq TM Products For qpcr using SYBR Green I Detection 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. 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. 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, Mg 2+ 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). 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 nonspecific 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 Amplification curve Melting curve SYBR Premix Ex Taq TM II (Perfect Real time) PCR conditions: Optimal 2- step protocol 95 C 30 sec. } 1 cycle 95 C 5 sec. 60 C 30 sec. 40 cycles Roche Premix Non Specific amplification FastStart Universal SYBR Green Master PCR conditions: 95 C 10 min. } 1 cycle 95 C 10 sec 60 C 30 sec 40 cycles ABI Premix Non Specific amplification Fast SYBR Green Master PCR conditions: 95 C 20 sec. } 1 cycle Figure 1 Target: Human TFRC Template: Serial dillution of cdna (equivalent to HL60 cells total RNA 10 pg ng; 1 reaction each) or H 2 O (NTC, in duplicate) Instrument: Thermal Cycler Dice Real Time System Results /Conclusion Reaction 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] 94 C 10 sec 60 C 20 sec 40 cycles 8

10 Real Time PCR Premixes Template Human testis cdna corresponding to total RNA 1 pg 100 ng Target genes A : LSP1 (GC 68%) B : C16orf84 (GC 73%) Figure 2 Comparison 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. RR420 company A(1) company B Template Human testis cdna corresponding to total RNA 10 pg 100 ng Target PGK1 (GC 54%) Target C16orf84 (GC 73%) 40 RR RR Ct Ct E 00 primer dimer 1.E 02 1.E 04 A(1) E 00 1.E 02 1.E 04 out of range B template RNA pg template RNA pg RR820 company A(2) company C Template Human testis cdna corresponding to total RNA 1 pg 100 ng unspecific Target PLSCR3 (GC 67%) Target ATP5F1 (GC 45%) out of standard curve Eff. 9.4 Eff. 94 Eff. 77 Eff. 88 Ct 30 Ct RR820 elongation time 30 sec.(2 step) reaction time 1 h 25 min A(2) 60 sec.(2 step) 2 h 1 min. 1.E 00 1.E 02 1.E 04 1.E 00 1.E 02 1.E 04 C 30 sec.(3 step) 2 h 13 min. template RNA pg template RNA pg Figure 3 Comparison 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. 9

11 Real Time PCR Premixes Premix Ex Taq (Perfect Real Time)(RR039A) For qpcr using TaqMan Probe Detection Method 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. Takara Premix Ex Taq (Perfect Real Time) Amplification Curve Applied Biosystems TaqMan Universal PCR Master Mix Amplification Curve 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. Standard Curve Takara Premix Ex Taq (Perfect Real Time) PCR conditions: 95 C 10 sec. } 1 cycle Standard Curve ABI s TaqMan Universal PCR Master Mix PCR conditions: 95 C 10 min. } 1 cycle Application an Gene Expression Assays (Applied Biosystems) Template: cdna seriatarget: Human ACTB Probe/Primer: TaqMl dilution (corresponding to total RNA 1 pg ng) 95 C 5 sec. 40 cycles 60 C 34 sec. (Approx. 50 min.) 95 C 15 sec. 40 cycles 60 C 1 min. (Approx. 90 min.) Results /Conclusion Takara 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] 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

12 Gene expression analysis PrimerArray TM Series 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 Taq TM Easy Analysis: With a tool exclusive for use with PrimerArray TM products. 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. 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. Steps for Preparation 1). 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 For Test Sample Master mix Master mix 4). Add primers (approx. 5 min): Add primers from the PrimerArray plate to the real-time PCR plate using a multichannel pipette, etc. PrimerArray For Control Sample For Test Sample 11

13 Gene expression analysis 5). Perform Real-time PCR (1-2 hours): The reaction is carried out with a real-time PCR instrument. Control Sample Test Sample 6). Data analysis is done using the PrimerArray Analysis Tool (about 30 min): Relative quantification analysis is performed using the ΔΔ Ct method. Scatter Plot 3D Profile PrimerArray TM Analysis Tool and How to use it 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 system Microsoft Office Excel 2003 Microsoft Office Excel 2007 The PrimerArray Analysis Tool Ver. 2.0 is available for download from the ara Bio Website. 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. tool-v2_en.pdf Scatter Plot The 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. 12

14 Gene expression analysis 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. Push this button to lead you to KEGG pathway Visualize the Expression Level Changes using the 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 Link to KEGG pathway (from the PrimerArrayTm analysis software) View of 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. 2 13

15 Licences 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 ,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. 14

16 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 rxns for Real Time (Mouse) EASY Dilution Solution x 1 ml Product name Product No. Quantity 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 rxns Amplification Kit (Real Time) Cell Amp Direct RNA Prep rxns Kit for RT-PCR (Real Time) Cell Amp Direct Kit for RT-PCR rxns (Real Time) & Protein Analysis * Larger sizes (with references LR and WR) already added with ROX reference Dye are also available PrimerArray TM Series products Product name (Human) Product No. Quantity PrimerArray TM Cytokine-cytokine PH001 1 set* receptor interaction PrimerArray TM Cell Cycle PH002 1 set* PrimerArray TM Cell adhesion molecules PH003 1 set* PrimerArray TM Jak-STAT signaling pathway PH004 1 set* PrimerArray TM Natural Killer cell PH005 1 set* mediated cytotoxicity PrimerArray TM Axon guidance PH006 1 set* PrimerArray TM Focal adhesion PH007 1 set* PrimerArray TM T cell receptor PH008 1 set* signaling pathway PrimerArray TM TGF-beta PH009 1 set* signaling pathway PrimerArray TM Wnt signaling pathway PH010 1 set* Product name (Mouse) Product No. Quantity PrimerArray TM Cytokine-cytokine PN001 1 set* receptor interaction PrimerArray TM Cell Cycle PN002 1 set* PrimerArray TM Cell adhesion molecules PN003 1 set* PrimerArray TM Jak-STAT signaling pathway PN004 1 set* PrimerArray TM Natural Killer cell PN005 1 set* mediated cytotoxicity PrimerArray TM Axon guidance PN006 1 set* PrimerArray TM Focal adhesion PN007 1 set* PrimerArray TM T cell receptor PN008 1 set* signaling pathway PrimerArray TM TGF-beta PN009 1 set* signaling pathway PrimerArray TM Wnt signaling pathway PN010 1 set* * enough reagent for 12 plates Recommended qpcr related products from Clontech Product name Product No. Quantity qpcr Human Reference Total RNA ug qpcr Human Reference cdna, oligo dt rxns qpcr Human Reference cdna, oligo dt rxns qpcr Human Reference cdna, random primed rxns qpcr Human Reference cdna, random primed rxns New from Clontech qpcr direct from tissue or dirty samples Product name Product No. Quantity Terra qpcr Direct SYBR Premix rxns Terra qpcr Direct SYBR Premix rxns For GC rich template / gdna /epigenetic SYBR Advantage GC qpcr Premix rxns Contact Information: Takara Bio Europe Website: orders@takara-bio.eu Tel: +33.(0) Fax: +33.(0) ZZWTBE1103EU