Improving cdna yield using SuperScript III. Debra Ann Nickson Senior Product Manager-Genomics qpcr Symposium Leipzig March 2005

Transcription

1 Improving cdna yield using SuperScript III Debra Ann Nickson Senior Product Manager-Genomics qpcr Symposium Leipzig March 2005

2 Overview Researchers needs for qrt-pcr Properties of SuperScript III reverse transcriptase RNA preparation Efficiencies of cdna synthesis measured in two-step qrt-pcr Choosing the best qrt-pcr product for your application One step vs two step Summary 2

3 Researchers needs for real-time qrt-pcr Dynamic range Broad linear range routinely over 6-7 orders of magnitude Sensitivity Low limit of detection (a few copies, sub-picogram levels of DNA/RNA) Specificity Quantify only the target of choice Multiplexing Detect multiple genes in one tube, commonly for amplifying gene of interest and endogenous reference gene simultaneously Convenience Closed tube assay format, mastermix configuration, amenable to high throughput applications Flexibility Compatible with various detection platforms and instrument platforms 3

4 X Thumb X Palm SuperScript III RT Engineering Three point mutations = SuperScript II RT RNase H Template-Primer Fingers X X Seven point mutations = SuperScript III RT Fidelity: MMLV, SuperScript II/III, AMV: ~ 5X10-5 error rate (LacZ assay) SuperScript III the new gold standard 4

5 RNaseH activity TTTTTTT AAAAAA TTTTTTT AAAAAA RNAse H TTTTTTT AA RNAse AAAA H reduces yield & full-length cdna RNAse H RNAse H TTTTTTT AAAAAA MML-V SuperScript III Total cdna 381ng 725ng Full length cdna 181ng 355ng Polymerase activity(units/mg) 330, ,000 Relative results using 2 µg of a 7.5kb RNA (40% which is poly A+) 5

6 SuperScript TM III Thermostability 50ºC Remaining RT Units (%) 100% 80% 60% 40% 20% 0% 50 0 C SuperScript III SuperScript II In cu b a tio n tim e (m in ) Reverse Transcriptase Half-life (min) at 50 C 55ºC Remaining RT Units (%) 100% 80% 60% 40% 20% 0% 55 0 C SuperScript III SuperScript II M-MLV Incuba tion Tim e (m in) SuperScript TM III SuperScript TM II MMLV ºC Remaining RT Units (%) 100% 80% 60% 40% 20% 0% 60 0 C SuperScript III SuperScript II Incuba tion Tim e (m in) 6

7 SuperScript III high cdna yields 7

8 SuperScript III Target Size Target Starting Total RNA (ng) 1X HML λ/hindiii fragments β-actin GAPDH 353 bp 532bp CBP 1.6 kb BF 2.4 kb HTF 3.6 kb VIN 4.6 kb GNE 5.9 kb ACTR 6.1 kb Pol ε 6.8 kb APC 8.5 kb FIB 9.4 kb Dynein 12.3 kb λ/hindiii fragments 1X HML dynein (12.3 kb) fibrillin (9.4 kb) adenomatous polyposis (8.5 kb) DNA polymerase ε (6.8 kb) nuclear receptor coactivator (6.1 kb) guanine nuc. exchange factor (5.9 kb) vinculin (4.6 kb) transcription factor (3.6 kb) B-factor properdin (2.4 kb) cap binding protein (1.6 kb) GAPDH (532 bp) β-actin (353 bp)

9 A word on template preparation RNA Template Preparation: Ready to use solutions TriZOL Silica-based technology PureLink Micro-to-Midi Total RNA Kit High-Throughput Isolation PureLink 96 Total RNA Purification Kit Eliminate genomic DNA contamination RNA should be treated with DNase I Amplification grade DNase I is then heat-inactivated by adding EDTA followed by incubation at 65ºC for 10 min. All RNA used should have an A 260 /A 280 ratio higher than

10 Efficiencies of cdna synthesis measured in two-step qrt-pcr 10

11 Comparison of two step RT-PCR Kits 10 ABI Taqman Gold RT-PCR Kit IVGN Real-Time 2 Step RT-PCR Kit B-Actin Taqman Primers 50 ng to 5 pg HeLa cdna Rn Cycle Number 11

12 Comparison of two step RT-PCR Kits y = (x) R 2 = Threshold Cycle (Ct) y = (x) R 2 = Starting Quantity (cdna concentration) 12

13 Comparison of cdna sensitivity competitive audit ABI cdna and SSIII cdna Using ABI Mix 10 ABI cdna IVGN SSIII cdna Both Using ABI PCR Mix y = (x) R 2 = ABI cdna IVGN SSIII cdna Both With ABI PCR Mix y = (x) R 2 =

14 Comparison of cdna sensitivity competitive audit ABI and SSIII cdna Using Supermix UDG 1.00E+01 ABI cdna IVGN SSIII cdna Both Using SM-UDG Mix Rn 1.00E E Threshold Cycle y = (x) R 2 = ABI cdna IVGN SSIII cdna Both Using Supermix UDG y = (x) R 2 =

15 The best qrt-pcr product for your application High performance, real-time quantitative RT-PCR requires high performance enzymes and systems SuperScript III Reverse Transcriptase with increased thermostability and half-life for higher cdna yields and better specificity with gene-specific primers Platinum Taq DNA Polymerase with antibody-mediated hot-start technology for higher PCR specificity and yield with room temperature setup for faster, more convenient setup, without performance loss Easy-to-use qpcr supermixes, one-step and two-step qrt- PCR systems 15

16 Automatic Hot Start PCR Assembly Initial Template Denaturation Temperature Cycling 96 C, 30 s - 2 min Inactive Taq DNA Polymerase Fully Active Taq DNA Polymerase 16

17 Reactivation of Platinum Taq activity Platinum Taq is activated quicker and has more enzyme activity during the PCR 17

18 Choice of qrt-pcr assay formats Two-Step RT-PCR Separate conditions for cdna synthesis & PCR Flexible choice of primer Typically oligo(dt) and/or random hexamers are used Ideal for quantification of multiple genes from a limited number of RNA samples One-Step RT-PCR Highly defined conditions to support RT and Taq Requires gene specific primer Higher throughput (many RNA samples) Can use all the RNA from a small sample Ideal for quantification of 1 or 2 messages from a large number of RNA samples 18

19 Flexible two step qrt-pcr kits SuperScript III Platinum Two-Step qrt-pcr Kit For LUX and probe based detection systems SuperScript III Platinum Two-Step qrt-pcr Kit with SYBR Green For detection based on SYBR Green Kit Components: SuperScript III Enzyme Mix 2x RT Reaction Mix E. coli RNase H DEPC-treated water 50mM MgCl 2 20x BSA 5 mg/ml ROX Reference Dye Platinum Quantitative PCR SuperMix-UDG or Platinum SYBR Green qpcr SuperMix-UDG 19

20 Convenient detection options SuperScript III Platinum Two-Step qrt-pcr Kit with SYBR Green provides easy and convenient detection with SYBR Green I dye 20

21 Convenient detection options SuperScript III Platinum Two-Step qrt-pcr Kit Provides specific detection with LUX Fluorogenic Primers Provides sensitive detection with TaqMan Probes 21

22 Quantitative RT-PCR from cell lysates SuperScript III Platinum CellsDirect Two-Step qrt-pcr Kit SuperScript III Platinum CellsDirect Two-Step qrt-pcr Kit with SYBR Green

23 Single Cell Detection 23

24 Comparison of Standard and HTP method Standard Method RNA+Primer+dNTP 65 degrees for 5 HTP Protocol/MasterMix format Combine components for first-strand synthesis: Enzyme Mix, Reaction Mix, RNA Template Place on ice for > 1 Add 10x buffer, MgCl2, DTT, and RnaseOut Mix Gently 42 degrees for 2 Add 1ul SSIII/reaction 25 degrees degrees degrees 5 Add 1ul Rnase H/reaction 37 degrees degrees degrees degrees 20 Add 1ul Rnase H/reaction 37 degrees 20 ready to use cdna 24

25 Sensitive one step qrt-pcr kits SuperScript III Platinum One-Step qrt-pcr Kit For LUX and probe based detection systems SuperScript III Platinum One-Step qrt-pcr Kit with SYBR Green For detection based on SYBR Green Kit Components: One-Step Enzyme Mix 2X Reaction Mix ROX Reference Dye 20X Bovine Serum Albumin 25

26 One-Step qrt-pcr System Superior sensitivity with dual-labelled probes 26

27 Ultra Sensitive one step qrt-pcr kits RNA UltraSense One-Step Quantitative RT-PCR System Kit Components: RNA UltraSense Enzyme Mix RNA UltraSense 5 x Reaction Mix ROX Reference Dye 20X Bovine Serum Albumin 27

28 Ultra Sensitive one step qrt-pcr kit 28

29 Two step kits product summary Characteristics High performance and complete kit for 2-step real-time RT-PCR, for highly sensitive and specific detection and quanitation of RNA in gene expression profiling studies Applications 2-step real-time RT-PCR, using fluorogenic detection (TaqMan, LUX ) or SYBR Green detection Real-time quantitative RT-PCR results directly from cells Ideal for Researchers doing 2-step qrt-pcr that want best performing reagents for reproducibility and sensitivity Researchers currently buying separate reagents to do 2-step qrt-pcr Researchers that want high-through-put compatible cdna synthesis protocols 29

30 One step kits product summary Characteristics Easy to use; reduce contamination and human error Optimized enzyme blend of SuperScript III RT and Platinum Taq DNA Polymerase RNA UltraSense 2.5 more concentrated than other supermixes Applications One-step real-time RT-PCR, using fluorogenic detection (TaqMan, LUX ) or SYBR Green detection Ideal for Researchers that want best performing reagents for reproducibility and sensitivity Researchers that need the highest sensitivity amplification of ultra lowabundance RNA Researchers currently buying separate reagents to do 2-step qrt-pcr Researchers that want rapid, streamlined protocols 30

31 X Thumb X Palm SuperScript III RT Engineering Three point mutations = SuperScript II RT RNase H Template-Primer Fingers X X Seven point mutations = SuperScript III RT Fidelity: MMLV, SuperScript II/III, AMV: ~ 5X10-5 error rate (LacZ assay) SuperScript III the new gold standard 31