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RNA product portfolio

RNA product portfolio. 03 August 2011 G. Brett Robb Staff Scientist RNA Division robb@neb.com. RNA Research, Development and Production Staff at NEB Ipswich. Research. Brett Robb. Ryan Fuchs. Larry McReynolds. Jingmin Jin. Alexander Zhelkovsky. Production. App Dev. John Greci.

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RNA product portfolio

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  1. RNA product portfolio 03 August 2011 G. Brett Robb Staff Scientist RNA Division robb@neb.com

  2. RNA Research, Development and Production Staff at NEB Ipswich Research Brett Robb Ryan Fuchs Larry McReynolds Jingmin Jin Alexander Zhelkovsky Production AppDev John Greci Yan Xu Eric Cantor Bhairavi Jani Dongxian Yue Bo Wu Leo Formenoy

  3. Research & Development • Focus on development of core RNA tools to complete our standard toolkit. • Enables NEB push further into the RNA market and supply innovative enzymes and reagents in the long term • Remain actively engaged in basic RNA research. • Discovery • Connections to the research community

  4. Under the hood: Production and QI • Leo, Dongxian, Bo, John G. • Scale up for greater yields and higher purity • Apply more stringent QC’s across the board • Mike, Dennis • Very large scale for several products • Bryce, Fermentation • Move all products to RNaseI (-) strains • Move fermentations to high density • Derek, Shawn, Christine • Perform quality control comparisons with other vendors’ enzymes • Establish specifications for new QC assays • Overnight RNase • qPCR • Relevant activity assays • Buffer compatibilities Outstanding purity and quality

  5. Sample Information 9,763 respondents were mailed with ~1,600 were undelivered 10% response rate (834 responded, with 207 partial and 627 complete) Some drop off, with 530 answering all questions; equivalent to 6.5% response rate 85% respondents are currently carrying out RNA research 95% of respondents were in the USA 70% were in academia, 13% in hospitals and 8% in Biotech 29% of respondents are PhD/Grad students, 21% are P.Is and 17% are Post Docs RNA Biology – Market Research Survey

  6. RNA Biology – Market Audit • Total RNA biology market: ~$800M • RNA synthesis: ~$33M • RNA stability: ~$31M • Selective RNA degradation: ~$13M • ID of RNA modifications: ~$10M • All segments expected to continue to grow Target markets niche in nature, but growing….

  7. RNA Biology – Market Research Conclusions Keys to success for NEB…. • Albeit niche, a growing market with unmet customer needs exists for NEB’s planned portfolio of RNA enzymes • As these needs are varied and diverse, a catalog of multiple enzymes with well characterized applications offers the best way to meet these needs • Product quality and clear protocols remain the most important parameters of supplier and product choice; the same key parameters that lead to Ambion’s success! Reinventing these for today’s researcher could be the key to differentiation and ultimate success for NEB • Price sensitivity appears to be high, perhaps as a result of LIFE’s (Invitrogen + ABI + Ambion) dominance of the field • To get a customer to change suppliers, improved performance and quality, followed by price, are the keys to success; prices having to be at least 20% lower to drive any change behavior

  8. RNA Products: Overview Transcriptome analysis (NEB Next), Functional Genomics (ShortCutRNAi), and others.

  9. RNA Synthesis Existing Products • SP6 RNA Polymerase • T7 RNA Polymerase • HiScribe T7 Kit • Cap Analogs • NTP mix • Poly(A) polymerase • Poly(U) polymerase New in 2010/11 In vitro transcription kit for wider application range • T7 High Yield RNA Synthesis Kit – E2040S • Long and short transcripts • Incorporation of modified nucleotides • co-transcriptional capping RNA Capping • Vaccinia Capping System – M2080 • Enzymatic mRNA capping of in vitro synthesized RNA • RNA 5’-cap labeling

  10. Making RNA in vitro • Why? • RNA structure and function studies • Hybridization studies • Ribozyme biochemistry • Antisense or RNAi • Microarrays • Microinjection/transfection • Translation • RNA vaccines • CureVac • Argos • BioNTech

  11. T7 High Yield RNA Synthesis Kit • Flexible system • Incorporate modified NTPs • Dyes • Biotin • Other modifications • Co-transcriptional mRNA capping • Radio-labeling • High or low specific activity • Enzyme mix, transcription buffer, separate NTPs. • Unique formulation for increased stability and extended shelf life

  12. Capping mRNA in vitro – Vaccinia Capping System • Significance • mRNA processing and translation • Transcript stability • Nuclear transport of mRNA • System components • Recombinant 2-enzyme system that adds cap 0 structures to in vitro transcripts • Applications • In vitro translation and mRNA turnover • mRNA transfection and microinjection • Cap-labeling of mRNA • High-yield capping for mRNA vaccine studies

  13. GLucUncapped RNA Outstanding Transcript Quality from the T7 High-Yield RNA Synthesis Kit With and Without Capping GLuc Capped RNA (Vaccinia) GLuc Capped RNA (Cap Analog)

  14. Capped RNA has increased translation efficiency Capped RNA Synthesis Cap Analog? Cap Using Enzyme? Uncapped RNA Capped RNA Capped RNA Mammalian Cell Transfection Readout Reporter Activity

  15. Prolonged Transcription Reactions With Minimal Template RNA Synthesis Reaction 3ng Template DNA Incubate at 37°C At each time point the corresponding tube is transferred to -20°C to stop the reaction 16 hr 24 hr 40 hr Column Purification of RNA Quantification Gel Analysis Company A NEB 16 24 40 16 24 40 Incubation Time (hrs) Conclusion: The T7High Yield RNA Synthesis Kit (NEB) synthesizes RNA of better quality and higher yield as compared to the competitor’s IVT kit.

  16. Separation & Purification Existing Products • Poly(A)+ RNA reagents • Magnetic mRNA Isolation Kit • Oligo (dT)25-Cellulose • polyA Spin mRNA Isolation Kit • Oligo dT25 Magnetic Beads • Protein-based enrichment • p19 siRNA Binding Protein • p19 miRNA Detection Kit • Enzymatic depletion • DNase I • XRN1 Product highlight • P19 miRNA detection kit - E3321S • Rapid and sensitive detection of specific miRNAs • Alternative to Northern Blotting • RT and PCR free

  17. Using P19 to detect and quantify miRNA • P19 miRNA detection kit - E3321S • Rapid and sensitive detection of specific miRNAs • Alternative to Northern Blotting • RT and PCR free

  18. Amplification Existing Products • Reverse transcription • M-MuLV Reverse Transcriptase • AMV Reverse Transcriptase • ProtoScript M-MuLV First Strand cDNA Synthesis Kit • ProtoScript AMV First Strand cDNA Synthesis Kit • RT-PCR • ProtoScript AMV LongAmpTaq RT-PCR Kit • ProtoScript M-MuLVTaq RT-PCR Kit • Priming Upcoming releases • mRNA decapping and RACE • Protocols/Application notes

  19. RT: M-MuLV or AMV? M-MuLV • Go-to RT • Can make cDNA up to 9.8 Kb AMV • Robust at a broad temperature range • Can be used for templates requiring higher reaction temperature + - + - + - 9.8 kb RT temperature 4.7 kb 37°C 65°C 9.8 kb 1.1 kb 5.8 kb 0.9 kb • Both RTs are available as stand-alone enzymes or in master mix cDNA synthesis kits

  20. Manipulation & Characterization Existing Products • Ligation • T4 Rnl1 • T4 Rnl2 • T4 Rnl2tr • Adenylated DNA linker • Labelling and modification • Hen1 • PNK • CIP • AntP • Selective modification • 5’-Deadenlyase • Selective Nucleases • XRN-1 • RNase H • RNase HII • Exonuclease T New in 2010/11 • Ligation • T4 Rnl2tr K227Q – M0351S/L • Lowest possible background for linker attachment • DNA 5’-Adenylation kit – E2610S • Make pre-adenylated cloning linkers of any sequence • Labeling and modification • In vitro transcription kits • mRNA Capping • Selective modification • RNA 5’-Pyrophosphohydrolase (RppH) – M0356 • Removes PP from PPP-RNA

  21. T4 Rnl2 T4 Rnl1 or or Use of RNA ligases OH + PO4 OH + pCp OH + PO4 OH + PO4 • Add known sequence to 5’-or 3’-ends of unknown RNA • 3’-end label • Make a longer RNA • Label site specifically

  22. Applications of T4 RNA Ligases Library prep for HTSeq RNA labeling for array hyb T4 Rnl1 • Best possible ligations are a necessity for: • demanding applications • sequencing • sensitive applications • microarray labeling • limited sample inputs • modifications that hinder ligation

  23. 5’-DNA Adenylation kit for producing adenylated adapters • Based on Mth RNA ligase • Enzymatic 5’-adenylation of ssDNA adaptors for next generation sequencing library preparation • Simpler than chemical or other enzymatic adenylation reactions • One-step reaction • Single-stranded DNA input • Increased yield (>95%) • Reduced post-modification purification • No need for gel purification • Scalable from pmol to μmol range

  24. Improved T4Rnl2tr variant • Advantages: • lowest possible side ligation products for demanding applications • Now commercially available • Applications: • Ligate an adenylated DNA or RNA to any RNA 3’-end • Join a single stranded pre-adenylated oligonucleotide adapter to small RNAs for cDNA library creation. • Join a single stranded pre-adenylated oligonucleotide adapter to RNA for strand-specific cDNA library construction. • Label RNA 3’-ends using pre-adenylated labeled donors

  25. Use of RNA ligases

  26. Size Markers and Reagents Existing Products • Ladders and Markers • ssRNA Ladder • dsRNA Ladder • Low Range ssRNA Ladder • siRNA Marker • microRNA Marker • RNase Inhibitors • Hs placental RNase Inhibitor • Murine RNase Inhibitor • VRC 2010/2011 releases • RNase Contamination Assay Kit – E3320S • Contains 300mer internally fluoroscein-labeled transcript • Detects general RNase activity in solutions and reagents • RNase Free reagents • RNA loading Dye (2X) – B0363 • Denaturing formamide based • Ribonucleotide Solution Set – N0450S/L • Separate rNTPs to allow for co-transcriptional labeling and capping

  27. RNase Contamination Assay Kit • RNase-free reagents are critical for RNA experiments • This assay provides a sensitive, easy to use method for detecting RNase activity. • Enzymatic RNase activity • General RNase activity • Heavy metals • High pH • Results are easily visualized using standard equipment • Included 300mer fluorescein-labeled RNA can be used for other assays

  28. NEBNext RNA Existing Products • NEBNext products for next generation library prep from DNA. • Illumina compatible (set 1) • 454 compatible (set 2) • SOLiD compatible (set 3) http://www.nebnext.com 2010/2011 Releases • NEBNext mRNA • Transcriptome analysis • Non-directional • Feeds into downstream NEBNext Kits • Small RNA Kit • Also for directional mRNA • Second strand synthesis module • Second strand synthesis (dNTP free) reaction buffer • For incorporation of dUTP

  29. Resources RNA reagent publications by NEB Scientists • 5’-Adenylase for making pre-adenylated DNA adapters • http://nar.oxfordjournals.org/content/early/2011/06/30/nar.gkr544.long • RNA ligases – properties and uses • http://www.biomedcentral.com/1472-6750/11/72 • http://rnajournal.cshlp.org/content/16/12/2537.long • P19 – use for detecting miRNA • http://www.biotechniques.com/BiotechniquesJournal/2010/June/Protein-mediated-miRNA-detection-and-siRNA-enrichment-using-p19/biotechniques-296750.html • http://www.nature.com/nnano/journal/v5/n11/full/nnano.2010.202.html

  30. Questions? Concerns? Suggestions? G. Brett Robb Staff Scientist RNA Division New England Biolabs Inc. robb@neb.com • Tough technical question? • Opportunities for collaboration? • Customers developing new approaches and looking for enzymatic solutions? • Do you or your customers have suggestions for RNA products you’d like us to make

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