1 / 42

RNA Technical Training

RNA Technical Training. 06 February 2013 G. Brett Robb Senior Scientist RNA Division robb@neb.com. Market Attractiveness Grid. Market Attractiveness. NEB’s Business Strength. Market Size Annual growth rate Historical profit margin Competitive intensity. Current market share

gil-cohen
Download Presentation

RNA Technical Training

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. RNA Technical Training 06 February 2013 G. Brett Robb Senior Scientist RNA Division robb@neb.com

  2. Market Attractiveness Grid Market Attractiveness NEB’s Business Strength • Market Size • Annual growth rate • Historical profit margin • Competitive intensity • Current market share • Annual share growth rate • Brand reputation • Research & Product Capabilities and Capacity • IP position • Channel to market/Promotional effectiveness ~1 year range

  3. Product Development: Applying the Market Attractiveness Grid Market Attractiveness NEB’s Business Strength

  4. RNA Market Attractiveness Grid 100M 50M 200M 430M 70M 31M NEB’s current market share (4.1M) 216M Room to grow NEB RNA reagent sales

  5. RNA Products: Overview Transcriptome analysis (NEB Next), and others.

  6. Framing RNA Reagents

  7. RNA Synthesis Existing Products • SP6 RNA Polymerase • T7 RNA Polymerase • HiScribe T7 Kit • Cap Analogs • NTP mix • Poly(A) polymerase • Poly(U) polymerase NEB has a large suite of RNA synthesis products 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

  8. Making RNA in vitro • Why? To make reagents and substrates for eperiments: • RNA structure and function studies • Hybridization studies • Ribozyme biochemistry • Antisense or RNAi • Microarrays • Microinjection/transfection • Stem cells/cell fate • Translation • RNA vaccines • CureVac • Argos • BioNTech

  9. 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 months vs competitor 6 months) • HiScribe™ T7 In Vitro Transcription Kit (E2030S) is a master mix set for production of unmodified RNA

  10. 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

  11. 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)

  12. 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 http://www.jove.com/video/3702/in-vitro-transcription-and-capping-of-gaussia-luciferase-mrna-followed-by-hela-cell-transfection

  13. 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.

  14. mRNA Capping • Which cap analog do I use? When? • 3´-0-Me-m7G(5')ppp(5')G RNA Cap Structure Analog – ARCA (S1411) • Highest incorporation rates of analogs • m7G(5')ppp(5')G RNA Cap Structure Analog (S1404) • Produces natural caps • m7G(5')ppp(5')A RNA Cap Structure Analog (S1405) • For capped transcripts where the first encoded base is A (using T7 Phi 2.5 promoter) • G(5')ppp(5')G RNA Cap Structure Analog (S1407) • For producing unmethylated caps • G(5')ppp(5')A RNA Cap Structure Analog (S1406) • For producing control A-capped transcripts When to use cap analogs vs. VACV capping? • VACV for ‘natural’ caps • Highest capping efficiency ARCA

  15. RNA Synthesis Business • NEB can provide: • OTC kits and individual reagents for research scale RNA synthesis • Bulk components to support customer synthesized RNA up to gram scale • Potential RNA synthesis service up to gram scale. • We are looking for customers for this business, and have capability

  16. cDNA Synthesis and RT PCR Existing Products • Reverse transcription • ProtoScript II M-MuLV RNaseH-Reverse Transcriptase • M-MuLV Reverse Transcriptase • AMV Reverse Transcriptase • First Strand cDNA Synthesis Kits • RT-PCR • ProtoScript AMV LongAmpTaq RT-PCR Kit • ProtoScript M-MuLVTaq RT-PCR Kit • OneTaq RT-PCR Kit • Priming

  17. RT Priming – Which priming strategy should I use? • Oligo d(T)23VN – S1327 • cDNA from poly(A)+ RNA only • Oligo d(T)18 – S1316 • cDNA from poly(A)+ RNA only • Random Primer 6 – S1230 • cDNA from all RNA • Random Primer 9 – S1254 • cDNA from all RNA • Random Primer Mix – S1330 • Contains Random 6 + Oligo d(T)23VN • cDNA from all RNA • Use this for most RT reactions • Oligo d(T)23VN – S1327 • Oligo d(T)18 – S1316 • Random Primer 6 – S1230 • Random Primer 9 – S1254 • Random Primer Mix – S1330

  18. ProtoScript II (M-MuLVRNaseH-) SuperScript II ProtoScript II • Performance = SSII • cDNA length • Elevated temperature

  19. RT: M-MuLVRNaseH- - cDNA product length M-MuLV RNaseH- • cDNA up to 12 kb in standard reactions

  20. RT: M-MuLVRNaseH- - Sensitivity • Detection of GAPDH RNA in as little as 1 pg of total RNA • 1 mammalian cell has 10-30 pg of RNA

  21. Which RT should I use?

  22. RNA Modification Enzymes– Where do they fit? RNA modifying enzymes are versatile tools that expand the capabilities of current RNA Seq and other analyses

  23. High throughput RNA sequencing is a transformative tool in RNA research mRNA Small RNA • Novel transcript discovery • RNA-protein interactions • RNA structural studies • Expression analysis • Genome annotation

  24. RNA modifying enzymes as tools that expand the capability of RNA analysis • Looking forward, RNA Seq will be used to ask increasingly refined questions in RNA research. This will require tools to: • Selectively capture some RNA species from a mixed population • mRNAs with modified bases • Capped mRNAs • Selectively represent some RNA species but not others in RNA Seq libraries • Selectively generate ligatable RNA ends • Selectively remove some RNAs • Nucleases • Affinity pullouts mono-phosphate di-phosphate tri-phosphate

  25. RNA Modifying Enzymes – current lineup Existing Products • Ligation • T4 Rnl1 • T4 Rnl2 • T4 Rnl2tr • T4 Rnl2tr K227Q • Labeling and modification • Hen1 • PNK • CIP • AntP • Selective modification • 5’-Deadenlyase • Selective Nucleases • XRN-1 • RNase H • RNase HII • Exonuclease T Recent releases • Ligation • T4 Rnl2tr KQ – M0373S/L • Lowest possible background for linker attachment • DNA 5’-Adenylation kit – E2610S • Make pre-adenylated cloning linkers of any sequence • Thermostable 5´ App DNA/RNA Ligase – (M0319S/L) • Ligate AppDNA to the 3’-end of ssDNA or RNA • Labeling and modification • mRNA Capping • Selective modification • RNA 5’-Pyrophosphohydrolase (RppH) – M0356 • Removes PP from PPP-RNA Competition

  26. RNA modifying enzymes as versatile tools that expand the capability of RNA analysis • Calf intestinal alkaline phosphatase and Antarctic Phosphatase • Phosphate removal for cloning • Phosphate removal before labeling • Selectively represent some RNA species but not others in RNA Seq libraries • Selectively generate ligatable RNA ends • T4 PNK • Phosphate addition for cloning • Phosphate addition for labeling • RNA 3’-end repair • Selectively represent some RNA species but not others in RNA Seq libraries • Selectively generate ligatable RNA ends mono-phosphate di-phosphate tri-phosphate

  27. RNA modifying enzymes as versatile tools that expand the capability of RNA analysis • RNA 5’-Pyrophosphohydrolase • Diphosphate removal from triphosphate ends leaves ligatable ends • Selectively represent some RNA species but not others in RNA Seq libraries • Selectively generate ligatable RNA ends • Xrn-1 • 5’-monophosphate-dependent 5’-3’ exonuclease • Selectively represent some RNA species but not others in RNA Seq libraries • Selectively remove some RNA species • rRNA depletion mono-phosphate di-phosphate tri-phosphate

  28. 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

  29. Design and bulk splicing of a covalently tagged pre-mRNA. RNA ligation enables site-specific RNA labeling. Crawford D J et al. RNA 2008;14:170-179

  30. Preparation and analysis of fluorescently labeled spliceosomesubcomplexes by CoSMoS. Hoskins AA et ak. Ordered and dynamic assembly of single spliceosomes. Science. 2011 Mar 11;331(6022):1289-95.

  31. 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

  32. Improved T4Rnl2tr variants K227Q and KQ • 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

  33. 5’-DNA Adenylation kit for producing adenylated adapters (E2610) • 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 5’-Phos DNA input • Increased yield (>95%) • Reduced post-modification purification • No need for gel purification • Scalable from pmol to μmol range

  34. Use of RNA ligases Common question: I want to ligate X to Y. Which ligase should I use? https://www.neb.com/tools-and-resources/selection-charts/rna-ligase-selection-chart

  35. 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 • RNase Contamination Assay Kit – E3320S • Contains 300mer internally fluorescein-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

  36. RNase Contamination Assay Kit Q) Your enzyme is contaminated with RNase A) No. It’s not. • 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 PK= Proteinase K added after incubation

  37. NEBNext RNA RNAseq: What's it for? • Transcriptome analysis • ID (new) transcribed regions • Gene expression profiling • mRNA • sRNAs • Refinement of gene models and annotation • Analysis of splicing • ID RNA regions bound by RNA binding proteins One Genome : Many transcriptomes • Cell, tissue or tumor type • Physiologic state • e.g. cytokine stimulated cells Current Kits • 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

  38. Resources RNA reagent publications by NEB Scientists • 5’-Adenylase for making pre-adenylated DNA adapters • http://nar.oxfordjournals.org/content/39/17/e117.long • RNA ligases – properties and uses • http://www.biomedcentral.com/1471-2199/13/24 • http://nar.oxfordjournals.org/content/40/7/e54.long • http://www.biomedcentral.com/1472-6750/11/72 • http://rnajournal.cshlp.org/content/16/12/2537.long • Use of enzymes in small RNA profiling • http://www.nature.com/nature/journal/v493/n7433/full/nature11716.html • http://www.hindawi.com/journals/jna/aip/360358/ • 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

  39. RNA Research, Development and Production Staff at NEB Ipswich Research Brett Robb Ryan Fuchs Larry McReynolds Jingmin Jin Alexander Zhelkovsky AppDev Production Yan Xu Ezra Schildkraut Bhairavi Jani Dongxian Yue Bo Wu Priscilla Hong Leo Formenoy

  40. Questions? Comments? Suggestions? G. Brett Robb Senior 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?

More Related