PrimeScript 1st strand cdna Synthesis Kit 1
Central Dogma replication transcription 0 processing translation 오늘의내용은? 조직추출 RNAiso Plus 5 mrna 우리가관심있는 mrna가차지하는비율이극히일부이기때문에관심있는 mrna를증폭해서그것을눈으로확인할정도로만들어주는것이다. 2
역전사반응 RT= Reverse transcription RT AAAAA TTTTT RT AAAAA TTTTT 역으로 ( 거꾸로 ) DNA 를합성 3
RT 반응에이용하는 primer AAAAA TTTTT XXXXXX XXXXXX ATGCTATGC XXXXXX AAAAA AAAAA 1. mrna 뒤에 poly(a) tail이붙는것을응용한 oligo(dt) primer: 분리한 RNA 중 mrna가모두 cdna로만들어진다 2. gene specific primer: target gene만 cdna로만들어진다. 3. 여기저기붙는 random primer: random primer( 주로 hexamer) 는염기서열이일정치않지만길이가고정된 primer이다. 이런 primer를넣고반응시키면길이도다양하고 random primer가붙은모든 RNA(rRNA, trna 포함 ) 가 cdna로만들어진다. 즉 cdna pool이만들어지는것이다. 4
One Step vs Two Step RT-PCR Two Step RT-PCR RNA Oligo(dT) 18 or random primers cdna RT buffer RT enzyme One Step RT-PCR RNA Gene-specific primers One-Step buffer PCR enzyme RT enzyme cdna dilution Few cdna sample PCR primers PCR buffer and enzyme PCR amplicon PCR amplicon Few RNA samples Many genes of interest Unlimited RNA (mgs) Many RNA samples Few genes of interest Limited RNA amount (ngs to pgs) 5
PrimeScript 역전사효소제품 PrimeScript Reverse Transcriptase M-MLV 에서유래한 High fidelity RTase 42 에서효율적으로역전사반응진행 long cdna 합성및 full length cdna library 제작에최적 Code Product Size Price( 원 ) Cost/rxns 비고 Promega A3802 ImProm-II Reverse Transcriptase 100 rxns 185,000 1,850 RT, buffer Life Technologies 18080044 SuperScrip III Reverse Transcriptase 10,000 U 410,000 8,200 RT, buffer TaKaRa 2680A PrimeScript Reverse Transcriptase 10,000 U 198,000 3,960 RT, buffer 6
PrimeScript kit 제품 PrimeScript 1st strand cdna Synthesis Kit * Kit 구성 (50 rxns) 1st strand cdna 합성에필요한 component 를모두갖춘 all-in-one kit 최고의역전사효소인 PrimeScript RTase 포함 반응의최적화로고차구조, 긴 RNA 적용가능 1. PrimeScript Reverse Transcriptase (200 U/ μl) 50 μl 2. 5 x PrimeScript Buffer 200 μl 3. RNase Inhibitor(40 U/ μl) 25 μl 4. dntp Mixture (10 mm each) 50 μl 5. Oligo dt Primer(50 μm) 50ul 6. Random 6 mers(50 μm) 100 μl 7. RNase Free dh2o 1 ml Code Product Size Price( 원 ) Cost/ml 비고 Life Technologies 18080-051 SuperScript III First-Strand Synthesis System 50rxns 561,000 11,120 Qiagen 205111 Omniscript RT Kit 50rxns 310,000 6,200 Promega A5000 GoScript Reverse Transcription System 50rxns 311,000 6,220 50 C 의역전사반응온도 PrimeScript 와비슷한 protocol 72 C RNA denature 온도 TaKaRa 6110A PrimeScript 1st strand cdna Synthesis Kit 50 rxns 279,000 5,580 TaKaRa 6210A PrimeScript II 1st strand cdna Synthesis Kit 50 rxns 290,000 5,800 7
PrimeScript RTase 를이용한 RT-PCR Kit 비교 PrimeScript RT-PCR Kit PrimeScript One Step RT- PCR Kit Ver.2 High Fidelity PrimeScript RT-PCR Kit 사용할수있는 RNA template 전반 DNA Polymerase Takara Ex Taq HS TaKaRa Ex Taq HS PrimeSTAR HS DNA Polymerase 증폭길이 12 kbp 의증폭을확인 8 kbp 의증폭을확인 6 kbp 의증폭을확인 특징 뛰어난신장성과매우높은증폭효율을갖는 2 step RT-PCR kit 역전사반응과 PCR 을연속실시하는 1 step RT-PCR kit 매우높은정확성으로 cdna 합성이가능한 2 step RT- PCR kit 8
Two Step RT PCR kit PrimeScript RT-PCR Kit High Fidelity PrimeScript RT-PCR Kit RR014A 50 회 285,000 원 R022A 50 회 407,000 원 * Kit 구성 (50 rxns) 1. PrimeScript RTase (for 2 step) 25 μl 2. 5 x PrimeScript Buffer 200 ul 3. RNase Inhibitor (40 U/ul) 25 ul 4. dntp Mixture (10 mm each) 150 ul 5. Oligo dt Primer (2.5 um) 50 ul 6. Random 6 mers (20 um) 50 ul 7. TaKaRa Ex Taq HS(5 U/ul) 25 ul 8. 10 x PCR Buffer II 250 ul 9. Control F-1 Primer (20 um) 10 ul 10. Control R-1 Primer (20 um ) 10 ul 11. Positive Control RNA 20 ul 12. RNase Free dh 2 O 1 ml * Kit 구성 (50 rxns) 1. PrimeScript RTase (for 2 step) 25 μl 2. 5 x PrimeScript Buffer 200 ul 3. RNase Inhibitor (40 U/ul) 25 ul 4. dntp Mixture (10 mm each) 50 ul 5. Oligo dt Primer (2.5 um) 50 ul 6. Random 6 mers (20 um) 50 ul 7. PrimeSTAR Max DNA Polymerase(2.5U/ul) 625 ul 2 8. 5 PrimeSTAR PCR Buffer 500 ul 9. Control F-1 Primer (20 um) 10 ul 10. Control R-1 Primer (20 um ) 10 ul 11. Positive Control RNA 20 ul 9
One Step RT PCR kit PrimeScript One Step RT-PCR Kit Ver. 2 * Kit 구성 (50 rxns) PrimeScript Rtase 와 Ex Taq HS 를조합한 1step kit 하나의 tube 에서 non-stop RT-PCR 가능 시약추가과정이없어조작이간편하고, 오염의위험을줄일수있음. 1. PrimeScript 1 step Enzyme Mix 100 μl 2. 2 1 step buffer 625 μl 2 3. Control F-1 Primer *1 (20 μm ) 20 ul 4. Control R-1 Primer *2 (20 μm ) 20 ul 5. Positive Control RNA (2 105 copies/ μl ) 20 ul 6. RNase Free dh2o 625 μl 2 Code Product Size Price( 원 ) Cost/ml 비고 QIAGEN 210210 QIAGEN OneStep RT-PCR Kit 25 rxns 224,000 8,960 RT, buffer mix, dntp Life Technologies 10928034 SuperScript One-Step RT-PCR System 25 rxns 274,000 10,960 RT, Buffer mix TaKaRa RR055A PrimeScript One Step RT-PCR Kit Ver. 2 50 rxns 384,000 7,680 RT, Buffer mix 10
One Step RT PCR Kit 비교 Maker Takara Qiagen Life Technologies Product PrimeScript One Step RT-PCR Kit Ver. 2 QIAGEN OneStep RT-PCR Kit SuperScript One-Step RT-PCR System RR055A 210210 10928034 용량 50회 25회 25회 가격 384,000 274,000 224,000 반응단가 7,680 10,960 8,960 Contents *PrimeScript One Step Enayme mix (PrimeScript RTase, Ex Taq HS, Rnase Inhibito r) *2 x One Step buffer (reaction buffer, dntp mixture, 1 step *Enhan cer Solution) *Control RNA, Control Primer *RNase Freee dh2o * RT-PCR Enzyme Mix (Omniscript Rtase, Sensiscript Rtase, HS Ta q DNA Polymerase) *5 X RT-PCR Buffer *dntp Mix *5 x Q-Solution *DNase-free water *Superscript III RT/ Platinum Taq Mix *2 X Reaction mix (reaction buffer, dntp mixture, MgSO4) *5mM Magnesium Sulfate Reaction *50 C 30min(RT 반응 ) *94 C 2min (RTase inactivation 반응 ) *PCR (25~30 cycles) *50 C 30min(RT반응 ) *45~55 C 15~30min(RT반응 ) *95 C 15min (RTase inactivation 및 chemic *94 C 2min (RTase inactivation 반응 ) ally modified HS Taq activation 반응 )) *PCR (35~40 cycles) *PCR (25~40 cycles) Method Buffer+ RNA+Enzyme+ Primer set Buffer+ RNA+Enzyme+ Primer set +dntp mix+ RNase inhibitor Buffer+ RNA+Enzyme+ Primer set + RNase inhibitor 차이점 *dntp 별도첨가과정불필요 *dntp 별도첨가과정필요 *RNase inhibitor가 enzyme mix에포함되어있 *RNase inhibitor 별도구매음 *Taq activation 과정필요 *dntp 별도첨가과정필요 *RNase inhibitor 별도구매 11
Real Time PCR 2013. 1. 22 12
PCR 원리 1 Cycle Heating Cooling Extension 95 55 72 Cycling Primer Polymerase Target DNA Cycling
PCR 원리? 이론적그래프 1 Cycle 2 Cycle 실제그래프 3 Cycle N Cycle
일반 PCR 의단점 Plateau Stage (End-point) End-Point PCR 은정량분석이불가능하다
일반 PCR 의단점 Agarose Gel 을이용한전기영동분석의한계는? 낮은정밀도낮은특이성주로크기에따른분류에사용실험자에의한오류가능성높음교차오염결과값을수치화하기어려움
Real Time PCR Growth curve Fluorescence Threshold Threshold cycle, C t 17
Real Time PCR 에사용하는방법 real-time PCR 은 PCR products 를형광을통해검출한다. 형광검출법은여러 가지가있으며, 그중에서가장대표적인것이 SYBR Green I 을이용한 검출법과형광 Probe 를이용한검출법이다.
SYBR Premix Ex Taq (Tli RNaseH Plus) Intercalator 를이용하는방법 (SYBR Green I)
Premix Ex Taq(Probe qpcr) 형광표지 probe 를이용하는방법
Real Time PCR 방법 Probe 방법 SYBR Green 특이성 SYBR보다높다 Probe 방법보다낮다 다중검정 가능 - Melting Curve - 가능 사용상용이성 까다롭다 쉽다 비용 비싸다 싸다 각방법별대표적인다카라제품코드 Probe RR390 SYBR RR820 RR420 21
SYBR 의단점을보완하기위한 Melting Curve
Real Time PCR 하려면 Template 조제 cdna 합성 Real Time PCR 반응
qpcr 효소선택가이드의변화 Sensitivity Fast qpcr Perfect RealTime PCR Primer 디자인 SYBR Premix Ex Taq (Tli RNaseH Plus) <Code RR420> SYBR Premix Ex Taq II (Tli RNaseH Plus) <Code RR820> SYBR Premix DimerEraser (Perfect Real Time) <Code RR091> Specificity 제품추천가이드 - qpcr 실험을처음하는고객에게첫번째추천시약 RR820 - 민감도 /Fast/ 긴단편의 qpcr 실험에 RR420 을추천 - RR420, RR820 보다높은반응특이성을원하는고객에게 RR091 24
Tli RNase H 기능이란? Takara qpcr Premix 의정확한정량 : Tli RnaseH 활용 타사제품에는없는오직다카라제품에만있는강점 - 특화된 Tli RNaseH 기능홍보 25
Tli RNaseH 효과 RR081 RR820 Tli RNaseH 가들어있는 RR820 에서 Ct 값이더낮게나왔다. Ct 값 : RR820<RR081 RR081+RNaseH RR081 에 RNaseH 를 처리하였더니 RR820 Ct 값이같았다. Ct 값 : RR820=RR081 26
RR820 vs RR420 RR420 YWHAZ (38%) RR820 X X RR820 is superior to RR420 for specificity. RR820 restrains the formation of primmer dimmers more than RR420. 27
RR820 vs RR420 RR420 Extension: 30s (TP900) ACTB : 186bp RR820 ACTB : 381bp ACTB : 533bp RR420 is superior to RR820 as for extension rate. 28
Takara vs Power SYBR(AB) ACTB : 186bp ACTB : 381bp ACTB : 533bp RR420 and RR820 are faster than PowerSYBR.
Takara vs Power SYBR FAST(AB) RR420 RR820 FastSYBR ACTB : 186bp X ACTB : 381bp X Although RR420 is as fast as FastSYBR, RR420 is more specific than FastSYBR. ACTB : 533bp X RR420 allows accurate quantification even for more long targets. 30 30
RR820 과최상의조합 PRTSS 실험결과향상을위한 tool TAKARA Perfect Real Time Support System Human, rat, mouse, cow, dog, chicken의 Refseq등록유전자에대한프라이머서열보유 온라인으로간편하게검색 Takara 제품과는반응조건의최적화과정불필요 http://takara.co.kr/web01/product/customservice02.asp
제품선택가이드 qpcr 시약선택가이드 폭넓은타겟에서특이성높은스탠다드시약 SYBR Premix Ex Taq II(Tli RNAseH Plus) (Code RR820A) 길이가다소긴타겟증폭이나고속모드기기적용 SYBR Premix Ex Taq (Tli RNAseH Plus) (Code RR420A) Primer dimer 등비특이적인증폭이있거나반응특이성을극대화할경우 SYBR Premix DimerEraser (Perfect Real Time) (Code RR091A) 게놈 DNA 또는 CpG islands 영역을증폭할경우 SYBR Premix Ex Taq GC (Perfect Real Time) (Code RR071A) 32
RR071 vs RR820, 420 Template : Human genomic DNA, Target : TFRC 38 36 34 32 Ct 30 28 26 24 22 20 0.0 5.0 10.0 15.0 20.0 Log2 (Template RNA pg) RR071 RR420 RR820 RR071 is superior to RR420 and RR820, using genomic DNA as a template. 33
RR071 vs 경쟁사 40 35 30 25 20 0.0 10.0 20.0 RR071 PowerSYBR 40 35 30 25 20 0.0 10.0 20.0 RR071 40 35 30 SYBRGreenE R 25 20 0.0 10.0 20.0 RR071 FastSYBR 40 35 30 25 20 0.0 10.0 20.0 RR071 KAPA 40 35 30 25 20 0.0 10.0 20.0 RR071 Quantitect 40 35 30 25 20 0.0 10.0 20.0 RR071 FastStart 40 35 30 25 20 0.0 10.0 20.0 RR071 QuantiFast Template : Human genomic DNA, Target : TFRC RR071 is suitable for quantification using genomic DNA as a template. 34
경쟁사별시약 브랜드제품명 Code 용량시장공략무기소비자가격 Bio-Rad itaq Universal SYBR Green Supermix 172-5121 5ml 기기, 인지도 300,000 IQ SYBR Green SuperMix 170-8880 2.5ml (Toyobo연계) 300,000 ABI Fast SYBR Green master mix 4385612 5ml 기기, 인지도 419,000 Roche LightCycler 480 SYBR Green I Master 04673484001 5ml 기기 390,000 QIAGEN QuantiTect SYBR Green PCR Kits(400) 204143 5ml 기기 381,000 Stratagene Brilliant III Ultra-Fast SYBR Green QPCR Master Mix 600882 4ml 기기 380,000 TOYOBO SYBR Green Real-time PCR master mix QPK-201 5ml 가격, Bio-rad 연계 290,000 KAPA KAPA SYBR FAST Universal 2X qpcr Master Mix KK4601 5ml 가격 330,000 Takara SYBR Premix Ex Taq II(Tli RNAseH Plus) RR820 5ml 인지도, 성능, 가격 350,000 35
주요경쟁사 : Bio-rad 브랜드제품명시장공략무기소비자가격 Takara SYBR Premix Ex Taq (Tli RNaseH Plus) 350,000 SYBR Premix Ex Taq II (Tli RNaseH Plus) Tli RNaseH PRTSS 350,000 Bio-Rad itaq Universal SYBR Green Supermix 300,000 기기, 인지도 (Toyobo연계) IQ SYBR Green SuperMix 300,000 TOYOBO SYBR Green Real-time PCR master mix 가격, Bio-rad 연계 290,000 장점 단점 1. Tli RNaseH 기능포함 2. 매뉴얼에다양한기종 protocol 등재 3. 초기셋업시쉽게 primer design을할수있는 PRTSS 4. 다양한실험에적용가능한제품구비 1. 낮은 qpcr 기기인지도 Takara( Bio-rad 대비 ) 공략포인트 1. Tli RNaseH 기능부각 2. CFX 96 장비에대한 protocol 이있어초기셋업이용이 ( 그외 AB, Roche, Cepheid등의 protocol등재 ) 3. PRTSS에대한이점홍보 ( 쉬운 primer design) 36
주요경쟁사 : AB(Life Technologies) 브랜드제품명시장공략무기소비자가격 Takara SYBR Premix Ex Taq (Tli RNaseH Plus) 350,000 SYBR Premix Ex Taq II (Tli RNaseH Plus) Tli RNaseH PRTSS 350,000 AB Fast SYBR Green master mix 기기, 인지도 419,000 Takara(Life Technologies 비교 ) 장점 단점 1. Tli RNaseH 기능포함 2. 매뉴얼에다양한기종 protocol 등재 3. 초기셋업시쉽게 primer design을할수있는 PRTSS 4. 다양한실험에적용가능한제품구비 1. 낮은 qpcr 기기인지도 공략포인트 1. Tli RNaseH 기능부각 2. AB 7300/7500/7500Fast, StepOnePlus 장비에대한 protocol이있어초기셋업이용이 ( 그외 Bio-rad, Roche, Cepheid등의 protocol등재 ) 3. PRTSS에대한이점홍보 ( 쉬운 primer design) 4. Pre heating 불필요 (Anti body hot start 방식의 Takara 시약 ) 5. 저렴한소비자가격 37