Transcription

1 Vol. 5 No Pioneer in RNAi innovation RNAi RNAi and double-strand RNA RNA Interference Shedding light on RNAi DNA-Directed RNA Interference Silencing Genes with a Hairpin Take the RiboMAX TM Express for RNAi Produce Functional sirnas and Hairpin sirnas using the T7 RiboMAX TM Express RNAi System Knocking down p53 expression using RNA interference Silence Expression with Codebreaker Reagent A resource for large-scale RNA-interference-based screens in mammals sirna library high-throughput screening SeouLin Bioscience

2 Tomorrow Lab, It s MyLab. Vortex Mixer Fuge Vortex Intelli Mixer MySpin 6500 Centrifuge & Vortex Programmable Centrifuge & Vortex Mini 3D Shaker Multi Programmable 3D Shaker Orbital Shaker 2xMicroplate Shaker Rocker Mini Rocker Magnetic Stirrer Hot Plate Stirrer Stirring Water Bath Thermo-Shaker 2x Microplate Thermo-Shaker Dry-block Thermostat Cooling & Heating Block IR Concentrator

3 BIOTIMES No Seoulin Bldg Songnae-Dong Kangdong-Gu Seoul ,KOREA TEL FAX Pioneer in RNAi innovation RNAi RNAi and double-strand RNA RNA Interference Shedding light on RNAi DNA-Directed RNA Interference Silencing Genes with a Hairpin Take the RiboMAX TM Express for RNAi Produce Functional sirnas and Hairpin sirnas using the T7 RiboMAX TM Express RNAi System Knocking down p53 expression using RNA interference Silence Expression with Codebreaker TM Reagent A resource for large-scale RNA-interference-based screens in mammals sirna library high-throughput screening Comparison of Expression Profile from Small Sample Protocol and Standard Protocol by Affymetrix GeneChip Microarray Expression microarray reproducibility is improved by optimising purification steps in RNA amplification and labelling New Products & Announcements

4 PureYield TM Plasmid Midiprep System Ordering Information PureYield TM Plasmid Midiprep System A preps A preps A preps Figure 1. Yield of pgem plasmid(b) purified from increasing amounts of JM109 bacterial culture using two different lysate preparation methods. JM109 cells containing pgem plasmid were grown in LB media overnight. Lysates were created as described in Section III.B (standard) or Section IV (alternative) protocols, and cleared using vacuum purification (Section III.D). As the biomass (culture volume and cell number) increases, the alternative clearing method is recommended over the standard lysate protocol (Section III.B). Figure 2. Comparison of transfection efficiency of purified plasmid DNA prepared using different midiprep systems. HeLa cells were transfected with psv- gal (Cat.# E1081) in a 96-well plate format using 0.1 plasmid/well. Twenty-four hours after transfection, galactosidase activity (using the Beta-Glo Assay System Cat.#E4720) and cell density (using the CellTiter-Glo Luminescent Cell Viability Assay Cat.#G7570) were monitored with a Fluorstar luminometer. The experiments were performed in triplicate.

5 silentgene TM U6 Hairpin Cloning system T7 RiboMAX TM Express RNAi System TranSilent TM sirna vectors Codebreaker TM sirna transfection reagent psicheck TM -1,2 vector sirna library

6 Perfect Match pgem -T Easy Vector System GoTaq TM DNA Polymerase Wizard SV Gel and PCR Clean Up System pgem-t Easy Vector I GoTaq Wizard SV Gel & PCR 50 prep (A9281)

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9 New Products ELISA Make Simple! Make Cheaper! ELISA SCREENING SET Antibody, Standard, Assay Reagent Kit Coating Antibody Detection Antibody Standard Streptavidin HRP Substrate Solution Stop Solution Analyte Cat.# Sample Type Range Sample Size Size Human IFN ESS0002 Cell Culture Supernate 15-1,000pg/ml 100ul/well 5 plate Human IL-6 ESS0005 Cell Culture Supernate pg/ml 100ul/well 5 plate Human TNF ESS0001 Cell Culture Supernate 15-1,000pg/ml 100ul/well 5 plate Mouse INF ESS0020 Cell Culture Supernate 47-3,000pg/ml 100ul/well 5 plate Mouse MCP-1 ESS0004 Cell Culture Supernate 15-1,000pg/ml 100ul/well 5 plate Porcine TNF ESS0003 Cell Culture Supernate 31-2,000pg/ml 100ul/well 5 plate Primate TNF ESS0006 Cell Culture Supernate 15-1,000pg/ml 100ul/well 5 plate 2 ELISA MINIKIT Antibody, Standard kit Kit Coating Antibody Detection Antibody Standard Analyte Cat.# Sample Type Range Sample Size Size Mouse IFN KMIFNG Cell Culture Supernate ,000pg/ml Variable 40 plate Mouse IL-2 KMIL2 Cell Culture Supernate 31-2,000pg/ml Variable 40 plate Mouse IL-4 KMIL4 Cell Culture Supernate pg/ml Variable 40 plate Mouse IL-5 KMIL5 Cell Culture Supernate 31-1,000pg/ml Variable 40 plate Mouse IL-6 KMIL6 Cell Culture Supernate 62-2,000pg/ml Variable 40 plate Mouse TNF KMTNFA Cell Culture Supernate 200-6,500pg/ml Variable 40 plate

10 New Products Thermo peptide & sirna WEB : thermopeptide@seoulin.co.kr TEL : 02) FAX : 02) Thermo

11 RNAi 1. Pioneer in RNAi innovation

12 Pioneer in RNAi innovation Dr. Nicholas Ng, Regional Manager Promega Corporation Preface Prior : Lecturer in Biological Instrumentation (Canberra Institute of Technology?part time 92-95) Product Specialist, ITS Science and Medical Pte. Ltd, Singapore 92 Education Officer (Ministry of Education, Singapore 1983? 1991) Education : Ph.D. -John Curtin School of Medical Research, Australian National University, Australia (96) MBA (Australia, 1995) Master Of Science (Distinction) in Plant Biotechnology, University of London Diploma in Education, National University of Singapore 1983 Bachelor of Science, National University of Singapore 1982 Grants & Awards : Public Service Commission Merit Bursary (Singapore) 1978 Lee Foundation Study Award 1991 Equity & Merit Scholarship (Australian Government ) ANU Ph.D. Scholarship Affiliations : Member of Singapore Polytechnic Biotechnology Advisory Committee Member of Consultative Committee (Division of Bioengineering) National University of Singapore. Publications : Kean Wooi Ng, Patricia Ridgway, Donna Ruth Cohen and David John Tremethick (1997) The binding of a Fos/Jun heterodimer can completely disrupt the structure of a nucleosome. The EMBO Journal (1997) Vol.16 No, 8, pp

13 RNAi BIOTIMES 11

14 12

15 RNAi Introduction 1. RNAi and double-strand RNA 2. RNA Interference

16 RNAi and double-strand RNA genes & development 13: Phillip A. Sharp 1 Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts USA 14

17 BIOTIMES 15

18 References 16

19 BIOTIMES TBS-380 Mini-Fluorometer 4S Small Simple Sensitive Sensibly Priced 17

20 RNAi : RNA Interference Promega Note 83, p By Natalie Betz. Ph.D., Promega Corporation Abstract History Mechanism 18

21 BIOTIMES Figure 1. Proposed mechanism of RNAi. RNase III family dsrna-processing dsrna. RNase III dsrna sirna. sirna multicomponent nuclease complexes (RISC). RISC mrna sirna RNAs. Synthesis and Use of dsrna 19

22 RNA Silencing and Other Small RNAs RNAi and the Genome Applications and Future Directions Conclusions References 20

23 21 BIOTIMES

24 RNAi technology 1. Shedding light on RNAi 2. DNA-Directed RNA Interference 3. Silencing Genes with a Hairpin

25 Everything You Need for RNAi BIOTIMES Shedding light on RNAi The Use of Bioluminescent Reporter Genes for RNAi Optimization Promega Note 87, p.6-9 By Jolanta Vidugiriene, Ph.D., Thomas Yeager, Ph.D., Brian Almond, Ph.D., Denise Garvin, M.S., Bob Bulleit, Ph.D., and Doug Storts, Ph.D. Promega Corporation RNAi bioluminescent Introduction psicheck TM vector 23

26 Figure 1. Mechanism of action of the psicheck TM Vectors. psicheck TM vector? Figure 2. Target site selection for p53 gene using psicheck TM -2 Vector. Panel A. HEK293T cells were seeded into a 96-well plate at a density of 3,000 cells/well. Human p53 cdna was subcloned into the psicheck TM -2 Vector using the Sgf I and Not I restriction sites. Cells were transfected with 0.02 of psicheck TM -2 Vector:p53 and 0.08 of silentgene TM -2 U6 Hairpin Cassette (blue bars) or psilentgene TM -2 Basic Vector (red bars) per well. CodeBreaker TM sirna Transfection Reagent (Cat.# E5052) was used for transfecting DNA cassette constructs, and TransFast TM Transfection Reagent (o) (Cat.# E2431) was used for vector DNA transfections. shrnas were directed against 5 target sites of human p53 as controls. Perfectly complementary shrna or a 4-base mismatch shrna against Renilla luciferase was used. The data are compared to cells transfected with shrna against nonspecific sequence. Forty-eight hours posttransfection Renilla and firefly luciferase activities were measured using the Dual- Luciferase Reporter 1000 Assay System (Cat.# E1980). The data are shown as percent suppression of Renilla activity normalized to firefly luciferase activity in cells transfected with shrnas against different target sequence or the nonspecific shrna sequence. The data is the mean of 12 wells plus or minus the standard deviation. Panel B. Inhibition of p53 was performed in HEK293T cells and analyzed by Western blot. Cells were treated with transfection reagent alone (lane 1), with the psilentgene TM -2 Basic Vector to a nonspecific sequence (lane 2) or to a p53 target site 4 sequence (lane 3). Transient assays were examined after 72 hours. psicheck TM vector 24

27 Figure 3. Measurement of changes in Renilla luciferase activity in live cells. HEK293T cells were plated in a 96-well plate at 3,000 cells/well. After an overnight incubation cells were treated with a transfection mixture consisting of 35 of serum-free medium, 0.3 of TransFast TM Transfection Reagent (Cat.# E2431), 0.02 of psicheck TM -1:p53 Vector and 0.08 of psilentgene TM -2 Basic Vector per well. For this experiment the psilentgene TM -2 Basic Vector expressed shrnas directed against human p53, Renilla or nonspecific sequence (negative control). After a onehour incubation, 100 of serum containing medium was added to the wells. At 17 hours post-transfection, EnduRen TM Live Cell Substrate (Cat.# E6481) was added to a final concentration of 60 M, and Renilla luciferase activity was monitored. BIOTIMES Figure 4. Screening of multiple potential shrnas. Panel A. The cdna for caspase-8 was cloned into the multiple cloning region of the psicheck TM -2 Vector creating psicheck TM -2:caspase-8 Vector. Three potential shrna were designed to specifically inhibit caspase-3, caspase-7, caspase-8 and caspase-9. The silentgene TM -2 Cassettes expressing shrnas were independently generated by PCR. The psicheck TM -2:caspase-8 Vectors were co-transfected with psilentgene TM -2 DNA cassettes against different targets into HeLa cells. At 48 hours post-transfection, Renilla and firefly activity was measured using Dual- Luciferase Reporter 1000 Assay System (Cat.# E1980). The Renilla luciferase relative light units were normalized to firefly luciferase expression. Panel B. psicheck TM Vectors into which cdna for caspase-3, 7, 8 or 9 had been cloned were co-transfected with silentgene TM -2 Cassettes expressing shrna to target sites designed for the corresponding caspase. At 48 hours post-transfection Renilla and firefly luciferase activity was measured as described in Panel A. The data are shown as percent suppression calculated based on the normalized Renilla activity in the cells transfected with nonspecific shrna to cells transfected with shrnas to different caspases and different target sites. 25

28 Conclusions shrna screening References 26

29 20/20 n Luminometer Superior Performance Sensitive The 20/20 n Luminometer provides exceptional sensitivity for all luminescent assays. Proprietary circuitry and an advanced photon-counting photomultiplier tube (PMT) produces unmatched signal-to-noise ratios. With a detection limit of 700 molecules of luciferase using Promega s Luciferase Assay Reagent, the 20/20 n is one of the most sensitive tube luminometers available. Flexible 8 Decades of Dynamic Range Flexible Sample Compartment Lid Start Injectors (Optional) Language Settings Features 3 x moles of luciferase using Promega s Luciferase Assay Reagent Linear dynamic range greater than nine decades Crosstalk better than 3 x 10-5 Single or dual injector (optional) Microplate 96-well format Easy to use software Veritas TM Microplate Luminometer Light Plate (optional) Easy to Use Microplate Luminometer The widest dynamic range enables the user to read both the dimmest and the brightest samples without multiple sample dilutions. This saves time and reduces errors. In some instances, it also saves precious samples. Complete interal auto-injection system integration ; Completely visible plumbing ; Veritas' open architecture enables the user to inspect all tubing and tips during flushing and priming. All reagent plumbing parts are clearly visible and easy to change or clean. Thus, problems can be seen and eliminated before a run is started. This feature saves valuable time, reagents and samples.

30 D Hairpin Cloning and Expression Made Easy NA-Directed RNA Interference Promega Notes 87, p.7~10 By Cheryl Bailey, Ph.D., Natalie Betz, Ph.D., Cindy Sprecher, B.S., Doug Storts, Ph.D., Jolanta Vidugiriene, Ph.D., and Thomas Yeager, Ph.D., Promega Corporation Abstract Introduction Figure 1. The mechanism of RNA interference. Double-stranded DNA (dsdna) is transcribed in vivo to produce shrna. In cells, double-stranded RNA sequences are recognized by an RNase III family member (e.g., Dicer in Drosophila) and are cleaved into short interfering RNAs of nucleotides. These sirnas are incorporated into an RNAi targeting complex known as RISC, which cleaves mrnas that are homologous to the integral sirna within the complex. 28

31 Figure 3. Easy detection of hairpin inserts by Pst I digestion. Ligation reactions containing the psistrike TM Vector and annealed hairpin oligonucleotides were transformed into JM109 cells, and individual colonies were selected. Plasmid DNA from individual colonies was digested with Pst I for 1 hour to determine the presence of hairpin insert. Lanes 2 10 of this 1% agarose gel shows the expected size fragments for successfully ligated hairpin inserts. Lane 1 is one of the uncut psistrike TM Vector constructs, and Lane M is the 1kb DNA Ladder (Cat.# G5711). BIOTIMES Figure 2. Overview of the sistrike TM U6 Hairpin Cloning System protocol. Target Site Selection Procedure for Cloning Hairpin Sequence 29

32 Transient In Vivo Suppression Figure 5. Suppression of p53 expression. 293T cells were transfected with two psistrike TM Vector constructs containing the same hairpin target sequence for p53 or a nonspecific target sequence. After 48 hours, cells were collected and lysed, and protein content was determined using the Pierce BCA assay. Either 1 or 2 of protein was loaded per lane and run on an 8% Tris-Glycine gel. The protein was then transferred to a nitrocellulose membrane and detected using monoclonal antibodies against p53 (Oncogene Research Products, Ab-2, 1:1,000 dilution) and a -actin loading control (Abcam, AC-15, 1:5,000 dilution). This was followed by detection with a secondary antibody, Goat Antimouse HRP (Cat.# W4021, 1:2,500 dilutions). The blot was visualized using Amersham ECL+ TM reagent and exposed to film. The amount of p53 protein was determined by densitometry. Figure 4. Transient suppression of Renilla luciferase with the psistrike TM - Basic Vector. Ten psistrike -Basic Vector constructs with the same Renilla luciferase hairpin target sequence (1 10) and a psistrike -Basic Vector construct with a nonspecific hairpin insert (NS) were created and transfected into HeLa cells stably expressing Renilla luciferase. At 48 hours post-transfection, cells were assayed for Renilla luciferase activity. Renilla luciferase activity was normalized to cell number, as measured by the CellTiter-Glo Luminescent Cell Viability Assay. Luminescence in cells with the Renilla luciferase-specific target sequence was then divided by the luminescence in cells with a nonspecific target sequence, and the results were expressed as the percent suppression. Figure 6. Stable suppression of Renilla luciferase with the psistrike TM - Puromycin Vector. CHO and HeLa cells stably expressing Renilla luciferase were transfected with the psistrike -Puromycin Vector containing hairpin oligonucleotides targeting Renilla luciferase or a nonspecific hairpin insert. Cells were selected with puromycin at a final concentration of 0.5 /ml for HeLa cells and 10 /ml for CHO cells. After 3 weeks, pools of selected cells were assayed for Renilla luciferase activity. Renilla luciferase activity was normalized to cell number, as measured by the CellTiter-Glo Luminescent Cell Viability Assay. Luminescence in cells with the Renilla luciferase-specific target sequence was then divided by the luminescence in cells with a nonspecific target sequence, and the results were expressed as the percent suppression. 30

33 Stable In Vivo Suppression Conclusion Protocol sistrike TM U6 Hairpin Cloning Systems Technical Manual #TM246,Promega Corporation. ( References 1. Duxbury, M.S. et al. (2004) Oncogene 23, Miyagishi, M. and Taira, K. (2002) Nat. Biotechnol. 20, Paddison, P.J. and Hannon, G.J. (2003) Curr. Opin. Mol. Ther. 5, Ordering Information Product Size Cat.# sistrike TM U6 Hairpin Cloning Systems Basic (a d) 1 system C7890 sistrike TM U6 Hairpin Cloning Systems Puromycin (a d) 1 system C7900 sistrike TM U6 Hairpin Cloning Systems Hygromycin (a d) 1 system C7910 sistrike TM U6 Hairpin Cloning Systems Neomycin (a d) 1 system C7920 For Research Use. (a) This product is covered under license from Carnegie Institution of Washington. Commercial use of this product may require a separate license from Carnegie. (b) Licensed under U.S. Pat. No. 6,573,099, Australia Pat. No and related patents from Benitec Australia, Ltd., and/or Commonwealth Scientific and Industrial Research Organisation. (c) Certain applications of this product are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used. (d) For nonprofit and noncommercial research use only. With respect to commercial research use or use by for-profit organizations, or any diagnostic or therapeutic uses, please go to for supply and licensing information. (e) The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. (f) U.S. Pat. No. 6,602,677, Australian Pat. No and other patents pending. sistrike and sicheck are trademarks of Promega. CellTiter-Glo is a trademark of Promega and is registered with the U.S. Patent and Trademark Office. BIOTIMES 31

34 S ilencing Genes with a Hairpin Introducing silentgene TM -2 U6 Hairpin Cloning Systems Promega Notes 87, p.11~14 By Jolanta Vidugiriene, Ph.D., Thomas Yeager, Ph.D., Cheryl Bailey, Ph.D., Cindy Sprecher, B.S., and Doug Storts, Ph.D., Promega Corporation Abstract Introduction Generating PCR DNA Cassettes Containing U6 Promoter and Hairpin shrna Target Sequences 32

35 Cloning a Hairpin Insert Into psilentgene TM Vectors BIOTIMES Figure 1. Overview of the silentgene -2 U6 Hairpin Cloning Systems procedure. The user provides the 5 phosphorylated, HPLC-grade downstream primer that contains the hairpin sequence specific for the target gene being studied. The upstream primer includes stop codons in all reading frames to facilitate blue/white selection. Figure 2. Amplification of hairpin structures. The silentgene TM U6 Cassette template was amplified using the silentgene TM U6 Cloning Upstream Primer, silentgene TM High Fidelity PCR Master Mix and 11 different downstream primers. All 11 primers generated the desired bp product. Amplification products were resolved using a 2% agarose gel (1X TAE, 0.5 /ml ethidium bromide). Lane M, 100bp DNA Ladder (Cat.# G2101). 33

36 Figure 3. Suppression of Renilla luciferase expression in HeLa cells by psilentgene TM Basic Vector. HeLa cells plated in 96-well plates were transfected with psilentgene TM Basic Vectors expressing shrna against the Renilla luciferase target site (two clones of this vector) or against a nonspecific sequence. Panel A. Forty-eight hours posttransfection, Renilla luciferase was quantitated by measuring luminescence after a 2-hour exposure of cells to EnduRen TM Live Cell Substrate (i,j) (Cat.# E6482). Panel B. Renilla luciferase activity was normalized to the relative amount of cells present in each well as determined using the CellTiter-Glo Luminescent Cell Viability Assay (k,l) (Cat.# G7571). Data are shown as averages of 6 replicates. Normalization of Renilla luciferase luminescence by cell number decreases the relative error of results. The average relative standard deviation decreased from 26% for the raw data in Panel A to 11% for normalized data in Panel B. Using psilentgene TM Vectors to Suppress Gene Expression 34

37 Conclusions BIOTIMES Figure 4. Stable suppression of p53 protein expression in HEK-293T cells. Cells were transfected with the psilentgene Puromycin Vector containing a p53-specific sequence (p53) or a nonspecific sequence (NS). Transient assays were examined 48 hours post-transfection. Pools of stably transfected cells were examined after 17 days of selection with puromycin while the puromycin-resistant clones were examined after 2 passages. Cells were collected, lysed and the proteins quantitated using the Pierce BCA assay. Either 1 or 2 of protein was loaded per lane and run on an 8% Tris-Glycine gel. The protein was then transferred to a nitrocellulose membrane and detected using monoclonal antibodies against p53 (Oncogene Research Products, Ab-2, 1:1,000 dilution) and a actin loading control (Abcam, AC-15, 1:5,000 dilution). This was followed by detection with a secondary antibody, Goat antimouse HRP (Cat.# W4021, 1:2,500 dilutions). The blot was detected using Amersham ECL+ reagent and exposed to film. Panel A. Western blot analysis showing shrna suppression of p53 expression in transfected cells. Panel B. Western blot analysis showing shrna suppression of p53 in established clones. Panel C. Expression of p53 in cloned cell lines was normalized to actin controls using scanning densitometry. (Control 293T = untransfected cells; transfection reagent = cells treated with transfection reagent only). References 35

38 In vitro transcription system 1. Take the RiboMAX TM Express RNAi 2. Produce Functional sirnas and Hairpin sirnas using the T7 RiboMAX TM Express RNAi System 3. Knocking down p53 expression using RNA interference

39 T The T7 RiboMAX TM Express RNAi System : Efficient Synthesis of dsrna for RNA Interference ake the RiboMAX TM Express for RNAi Promega Note 84, pg7-11 By Natalie Betz, Ph. D., and Tracy Worzella, B. S., Promega Corporation BIOTIMES Abstract Introduction Figure 1. Protocol for the production and purification of dsrna using the T7 RiboMAX TM Express RNAi System. 37

40 Template Considerations Figure 2. Native gel analysis of different-sized dsrnas generated using the T7 RiboMAX TM Express RNAi System. Approximately molecules of each dsrna were separated on a 1.8% agarose/1x TAE gel and visualized by staining with 0.5 g/ml ethidium bromide. Lane designations: lane 1, 74ng 180bp ERK-A dsrna; lane 2, 200ng 500bp Renilla dsrna; lane 3, 200ng 505bp ERK- A dsrna; lane 4, 312ng 778bp ERK-A dsrna; lane 5, 400ng 1,000bp Renilla dsrna; lanes M, 1kb DNA Ladder (Cat.# G5711). Note that dsrna migrates more slowly than doublestranded DNA markers. Figure 3. Comparison of dsrna yields from PCR and plasmid templates. PCR templates (either 100 or 500ng DNA) and an equimolar amount of plasmid template (500ng or 2.5 g) were used to produce dsrna for the same 778bp ERK-A target. Each reaction contained an equal mix of separate T7 forward and T7 reverse DNAs. The results are the average of duplicate reactions analyzed by agarose gel electrophoresis followed by SYBR Green II staining. RFU = Relative Fluorescence Units. dsrna Functional in RNAi 38

41 Figure 4. Western blot analysis of the ERK-A (MAPK) protein from S2 cells incubated in the presence or absence of ERK-A 778bp dsrna or Renilla 500bp dsrna. The blot was generated and analyzed as described in the Methods section. Incubation with the secondary antibody alone showed no bands (data not shown). Lane designations: lane 1, no dsrna (negative control); lane 2, 25 g ERK-A dsrna; lane 3, 25 g Renilla dsrna (nonspecific control). Results represent pooled triplicates for each treatment. BIOTIMES Conclusions Methods 39

42 References Protocols 40

43 Produce Functional sirnas and Hairpin sirnas using the T7 RiboMAX TM Express RNAi System Promega Note 85, pg15-18 By Natalie Betz, Ph.D., Promega Corporation BIOTIMES Abstract Introduction Figure 1. Protocol for producing and purifying sirna using the T7 RiboMAX TM Express RNAi System. Synthesizing sirna with the RiboMAX TM System 41

44 Figure 2. Native polyacrylamide gel analysis of different sirna molecules generated using the T7 RiboMAX TM Express RNAi System. Approximately 50ng of each sirna was analyzed on a 4-20% TBE PAGE gel with TBE running buffer. Following electrophoresis the gel was stained with 0.5 /ml ethidium bromide. Lane designations: lane 1, 10bp DNA Step Ladder (Cat.# G4471); lane 2, 21bp chemically synthesized Renilla sirna (Dharmacon); lane 3, 21bp Renilla sirna synthesized using the T7 RiboMAX TM Express RNAi System; lane 4, 21bp p53 sirna synthesized using the T7 RiboMAX TM Express RNAi System; lane 5, 49-nucleotide long Renilla shrna (19bp duplex with 9 nucleotide loop and 2-uridine 3 overhang) synthesized with the T7 RiboMAX TM Express RNAi System. Note: sirna migrates more slowly than double-stranded DNA. Template Design and Considerations Table 1. Yields of Six Different sirna Templates Synthesized Using the T7 RiboMAX TM Express RNAi System (average of triplicates per template). sirna Sample Yield (mg sirna/ml reaction) Renilla Site 1 sirna 0.85 Renilla Site 1 hairpin sirna 4.20 Renilla Site 2 sirna 1.50 Renilla Site 2 hairpin sirna 2.10 Renilla Site 3 sirna 1.20 Renilla Site 3 hairpin sirna 1.70 sirna Hairpin sirna RNAi 42

45 BIOTIMES Figure 3. Required DNA oligonucleotides to generate templates for making sirna or hairpin sirna. The sense sequence of the target mrna is assumed to be the protein coding sequence. Figure 4. Comparing three different Renilla sirnas generated either chemically or in vitro using the T7 RiboMAX TM Express RNAi System to reduce the expression of Renilla luciferase. CHO cells stably expressing the codon-optimized Renilla luciferase (hrluc) gene were transfected with 20ng of sirna to three different sites in the hrluc mrna. In addition, in vitro synthesized shrnas to the same 3 sites were also tested. As a negative control a scrambled sirna was used; as a positive control chemically synthesized sirnas were used (Dharmacon). Transfections were performed in replicates of 8 in 96- well plates using the silentgene TM Transfection Reagent. Following transfection (24 hours), quadruplicate wells were processed for either hrluc activity using the Renilla Luciferase Assay System (d,e) or cell number using the CellTiter-Glo Luminescent Cell Viability Assay (e,f). The ratio of the the average hrluc signal compared to the average CellTiter-Glo signal for each sirna transfection was calculated, and the decrease in this ratio relative to the scrambled sirna control was determined. The data is presented as the percent decrease in the Renilla/CellTiter-Glo Assay ratio as compared to a transfected scrambled sirna. D = chemically synthesized sirna from Dharmacon; P = in vitro transcribed using the T7 RiboMAX TM Express RNAi System; HP = shrna in vitro transcribed using the T7 RiboMAX TM Express RNAi System. All in vitro generated sirnas and shrnas were synthesized purified as described in Technical Bulletin #TB316. The sirnas and shrnas were quantitated by 2.5% agarose/1x TAE gel analysis and compared to known amounts of a chemically synthesized sirna. Following electrophoresis, the gel was stained with 1:10,000 SYBR Green II stain(molecular Probes) for 20 minutes and the gel scanned and quantitated using a Molecular Dynamics STORM fluorescent scanner (blue mode; PMT = 1,000). Figure 5. Suppression of endogenous p53 protein. Twenty-four hours after plating in a 12-well plate, 293T cells were transfected with 200ng scrambled sirna (lane 1), 200ng in vitro synthesized p53 sirna (lane 2), or 200ng chemically synthesized p53 sirna (lane 3) using the silentgene TM Transfection Reagent. Twentyfour hours following transfection cells were lysed using 1X Reporter Lysis Buffer (Cat.# E3971) containing protease inhibitors and the protein quantitated using the BCA Protein Assay (Pierce). Equal amounts of each lysate (10 ) were separated on a 4-12% polyacrylamide Bis-Tris gel (Invitrogen) and transferred to Hybond-C membrane (Amersham). The blot was probed with both a p53 antibody (Calbiochem) and a -actin antibody (Abcam). Detection was done using a goat anti-mouse HRP conjugate (Cat.# W4021) and the chemiluminescent detection reagents in the Transcend Chemiluminescent Non-Radioactive Translation Detection System (Cat.# L5080). The blot was exposed to Kodak X- OMAT film for approximately 4 minutes. In addition, the simultaneous detection of the -actin protein controlled for loading and transfer. The p53 and -actin bands are indicated and are of the expected sizes. 43

46 Conclusion References Ordering Information Product Size Cat.# T7 RiboMAX TM Express RNAi System 50 reactions P1700 silentgene TM U6 Cassette RNA Interference System 20 reactions C

47 K nocking down p53 expression using RNA interference Cell Notes Issue 7, p.12~13 BIOTIMES Introduction sirna Without Cloning silentgene TM System p53 knockdown 45

48 Figure 1. Suppression of endogenous p53 protein. 293T cells were transfected in a 12-well plate with 1 /well of U6 cassette (0.5 of each PCR product) and 6 /well of silentgene TM Transfection Reagent. The cells were then incubated for 72 hours, lysed and the protein quantitated. Five micrograms of protein/lane was run on a 10% tris-glycine gel and transferred to a nitrocellulose membrane. The membrane was probed with both a p53 (1:1,000 Calibiochem Ab-2) antibody and a -actin (1:5,000 Abcam, Ab 6276) antibody to serve as a loading control. A goat anti-mouse, horseradish peroxidase-conjugated secondary antibody was used followed by chemiluminescent detection. In the first lane, a nonspecific U6 cassette was tested as a negative control. The second lane was transfected with a p53-specific U6 cassette. Experiments were performed in duplicate. Figure 2. Suppression of endogeneous p53 protein. 293T cells were transfected with 200ng scrambled sirna (lane 1), 200ng in vitro synthesized p53 sirna (lane 2), or 200ng chemically synthesized p53 sirnas (lane 3). Equivalent amounts of each lysate (10 ) were separated on a polyacrylamide gel, transferred to Hybond -C membrane and probed with both a p53-specific antibody and a -actin antibody. Detection was performed using a HRP-conjugated secondary antibody and chemiluminescent detection reagents. Each lane represents the mean and average of duplicate wells per transfection. The p53 and -actin bands are indicated and are of the expected sizes. T7 in vitro sirna Summary 46

49 sirna reagent 1. Silence Expression with CodeBreaker TM Reagent sirna 1. A resource for large-scale RNAinterference-based screens in mammals 2. sirna library high-throughput screening

50 S ilence Expression with CodeBreaker TM Reagent CodeBreaker TM sirna Transfection Reagent : Efficient Trnasfection of sirna Duplexes into Mammalian Cells Promega Note 86, pg21-22 By Thomas yeager, Ph.D., and Jolanta Vidugirienne, Ph.D., Promega Corporation Abstract Introduction CodeBreaker TM sirna Transfection Reagent Figure 1. Flow diagram showing the transfection process using the CodeBreaker TM Reagent. 48

51 Figure 2. CodeBreaker TM sirna Transfection Reagent with various cell lines. CHO, 3T3 and HeLa cell lines stably expressing a humanized Renilla luciferase were created by cotransfection of a humanized Renilla expression construct(phrl-sv40 (a,b,c,d), Cat. # E6261) and a second construct containing a selectable marker. sirna transfections were performed using /well CodeBreaker TM Reagent and 2.5.6nM/well Renilla-specific sirna or a control nonspecific sirna. Twenty-four hours after transfection the cells were assayed for the reduction of Renilla luminescence in 96-well plates using 3,000 cells/well. The values represent mean percent suppression of Renilla activity by the specific sirna duplex S.D., n=10. Conclusion Protocol Additional Resources BIOTIMES Ordering Information Product Size Cat.# CodeBreaker TM sirna duplex Transfection Reagent 0.4 ml E ml E5053 T7 RiboMAX TM Express RNAi System 50 x 20ul reactions P1700 Figure 3. Suppression of p53 following transfection of p53-specific sirna using the CodeBreaker TM sirna Transfection Reagent. The day before transfection, 293T cells were plated at a density of 40,000 cells/well in a 12-well dish. Each well was transfected using 6 CodeBreaker TM Reagent and either p53- specific ornonspecific sirnas at a final concentration of 3nM. The cells were collected after 72 hours, and the protein was quantitated. Western blot analysis was performed using 1 of protein per lane. The proteins were detected using either a p53 monoclonal antibody (Oncogene Research Products, AB-2, 1:1,000) or an actin loading control (Abcam, AC-15, 1:5,000). A horseradish peroxidaseconjugated goat anti-mouse secondary antibody was used (Cat.# W4021, 1:2,500) followed by chemiluminescent detection. The resulting films were quantitated by densitometry. Lane 1: an 83% inhibition in levels of p53 synthesized was seen when compared to controls transfected with nonspecific sirna (lane 2). 49

52 A resource for large-scale RNA-interference-based screens in mammals nature 428: Patrick J. Paddison 1 *, Jose M. Silva 1 *, Douglas S. Conklin 1 * Mike Schlabach 2, Mamie Li 2, Shola Aruleba 1, Vivekanand Balija 1, Andy O'Shaughnessy 1, Lidia Gnoj 1, Kim Scobie 1, Kenneth Chang 1, Thomas Westbrook 2, Michele Cleary 3, Ravi Sachidanandam 1, W. Richard McCombie 1, Stephen J. Elledge 2 & Gregory J. Hannon 1 1 Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA 2 Department of Biochemistry,HowardHughesMedical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA 3 Rosetta Inpharmatics, th Avenue NE, Kirkland, Washington 98034,USA * These authors contributed equally to this work Present addresses: Department of Biomedical Sciences, Center for Functional Genomics, University at Albany, East Campus, B342A, One University Place, Rensselaer, New York , USA (D.S.C.); Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA (M.S., T.W. and S.J.E.) Figure 1. pshag-magic shrna cassette movement strategy. a, Map of the pshagmagic vector. CMr, chloramphenicol-resistance gene; Kanr, kanamycin-resistance gene; LTR, long terminal repeat; OriT, origin of transfer. b, A diagrammatic representation of DNA exchanges occurring once the pshag-magic donor vector has been transferred to cells containing a recipient vector by mating. In this case plenti-loxp (M.L. and S.J.E., unpublished data) is the recipient vector. WPRE, woodchuck hepatitis B posttranscriptional responsive element. c, Plasmids from ten independent colonies (post-mating) were digested with NdeI and the digestion products were separated on a 0.5% agarose gel (lanes 1-10). The parental plasmids, pshag-magic (lane 11) and plenti-loxp (lane 12), each contain a single NdeI site, and on cleavage generate a 7.0- or kilobase (kb) fragment, respectively. The pshag-magic vector contains a unique NdeI site in the U6 promoter, which is transferred into the recipient vector. The correct mating product generates two fragments of 3.5 kb and 7.4 kb. M, marker. 50

53 BIOTIMES 51

54 Figure 2. Microarray analysis of pshag-magic library bar codes. a, Selfself hybridization of pshag-magic library bar codes obtained from approximately 15,000 library plasmids prepared from E. coli. The DNA microarray (Agilent Technologies) is composed of 20,241 complementary 60- nuclotide oligonucleotides out of a total of 22,575 elements on the array, including controls; the remaining represent various positive and negative controls. Cy-labelled crna for library bar codes was generated and competitively hybridized as described in the Supplementary Methods. b, An analysis of a subset of nucleotide bar codes from a versus nucleotide shrna probes. Each 60-nucleotide shrna probe contains a direct repeat of the 29-nucleotide genetargeting sequence with 2-nucleotide spacer c, Miroarray abalysis of pshag-maglc bar codes from transduced NIH3 T3 cells. Two pools of 2 X 10 7 cells (A and B) were infected separately and harvested 48 h after infection. Bar codes were processed and competitively hybridized as described in the Methods. The x axis is an index of all the elements (22,575), bar codes, hairpins and controls on the array. The y axis shows the mean measured ratios from two experiments (forward and reverse colour orientation) of 3T3 pool A hybridized against 3T3 pool B, plotted on a log scale with the numbers indicating fold change. Most probes, with the exception of a few, scatter around 1.0 (no change) with a range between 0.5 (twofold decrease) and 2.0 (twofold increase). d, Microarray analysis of pshag-magic bar codes in transduced normal human diploid fibroblasts (IMR90) cells carried out as in c. Figure 3. A reverse genetic screen for defects in human proteasome function. a, A graph of relative ZsGreen fluorescence for all pshag-magic clones transfected. Red vertical bars indicate 22 positively scoring proteasome shrnas corresponding to 15 known proteasome subunits, whereas black bars indicate library shrnas. The yellow horizontal bar indicates a cut-off that was set based on control experiments. b, A replicate transfection experiment as in a using a set of 22 positively scoring proteasome hairpins and 33 that failed to score (blue bars), along with non-proteasome hairpin controls (green bars). These experiments were carried out in triplicate. All 22 of the proteasome hairpins with a positive score from the original screen were re-tested and also achieved a positive score. None of the 33 non-scoring proteasome hairpins from the original screen managed to match the score of the 22 positively scoring proteasome shrnas from the first round (yellow line). However, in this experiment 36 of 55 scored well above (,2 s.d.) the mean background (red line). c, Fluorescence microscopy images showing representative results for individual proteasome hairpins as carried out in a and b. A shrna targeting firefly luciferase (Ff-1) and small molecule proteasome inhibitor, MG-132 (Sigma), were used as negative and positive controls, respectively. d, A diagrammatic representation of the 26S proteasome colourcoded according to pshag-magic library hits. Subunits coloured green had strong positive shrna hits from the library in the primary screen. Grey subunits were not represented by any shrnas in the approximately 7,000 tested. Subunits coloured blue were represented by at least 1 shrna but did not score in the screen. For nomenclature see Supplementary Table

55 Methods Figure 4. Further validation of selected pshag-magic proteasome hairpins. a, Western blot showing specific suppression of the ATPase-1 and non- ATPase-1 of the 19S regulatory subunit in transiently transfected HEK293 cells. Cells were transfected with shrnas as indicated. Knockdown of protein levels for shrna-1 and -3 against ATPase-1 proteasome correlated with the severity of the relative scores in the pzsgreen-modc accumulation assay. The lane labelled untransfected indicates the control lane where cells were not transfected. b, A western blot showing increased steady-state levels of endogenous c-myc in HEK293 cells transiently transfected with library shrnas as indicated. c-myc is normally degraded by ubiquitin-mediated proteolysis in these cells19. BIOTIMES 53

56 References 54

57 10 bits Innovation GelDoc-It System BIOTIMES Ordering Information Product Cat.# GelDoc-It System(20x20, 302nm) x26, 302nm(Hi/Low) x20, 254/302/365nm x26, 254/302/365nm GelDoc-It Gel Camera Type: Specifications 1/2" Progressive Scan Interline CCD Monochrome 1.4 Megapixel (1360 x 1024) Capture Resolution: Pixel Size: Bit Depth: 10-bit Binning: 1x1, 2x2, 4x4, 8x8 for increased sensitivity PC Interface: FireWire (IEEE-1394) Optics Interface: C-Mount Optics: 6X manual zoom Darkroom Dimensions: 18.25"H 14"W 11"D (463 x 356 x 279 mm) Features: White light epi-illumination Ethidium Bromide interference filter UV protective gel viewer Transilluminator, 25 26cm filter, 302nm UV Options Transilluminators: PC: Printers: Converter Plates: Emission Filters: 3UV (254/302/365nm) or single UV, 20 20cm or 21 26cm. High specification configuration Thermal (256 gray scale) UV to White Light or UV to Visi-Blue 480 nm SYBR Green and SYBR Gold 55

58 SiRNA library high-throughput screening Transduction Treatment (optional) Figure 1. Construction of GeneNet TM sirna Libraries the sirna templates are synthesized with the ends that are ligationcompatible with the digested pfiv library vector. Specific details of the restriction enzymes, insertion sites, and the oligonucleotide designs vary from library to library and are provided with the documentation for the specific libraries. Following construction, each library is packaged into highly transducible VSV-G pseudotyped lentiviral particles by passage through packaging cells that are co-transfected with the pfiv-pack TM Lentiviral Packaging kit. All GeneNet Libraries are provided in a pre-packaged, transduction-ready, ready-to-use form. 56

59 Selection Amplification Identification BIOTIMES Figure 2. Using GeneNet TM sirna Libraries Functional gene screening with a ready-to-transduce packaged GeneNet Library involves the following steps GeneNet TM sirna library GeneNet TM library vector Target gene sirna Cat # 1.5K Human cancer pfiv-h1-copgfp 1500(cancer ) 7500 SI200A-1 1.5K Human cancer pfiv-h1-puro 1500(cancer ) 7500 SI203A-1 1.5K Human cancer pfiv-h1-copgfp 8500(cancer ) SI201A-1 1.5K Human cancer pfiv-h1-puro 8500(cancer ) SI202A

60 1. Comparison of Expression Profile from Small Sample Protocol and Standard Protocol by Affymetrix GeneChip Microarray 2. Expression microarray reproducibility is improved by optimising purification steps in RNA amplification and labelling

61 C omparison of Expression Profile from Small Sample Protocol and Standard Protocol by Affymetrix GeneChip Microarray Kyoung-Mun Lee, Soo-Moon Song and Hee-Kyung Song Seoulin Bioscience Institute, Seoulin Bioscience Co. Ltd., , Seoul, Korea BIOTIMES Abstract Introduction Materials and Methods Figure 1. Schematic Diagram of the Standard Protocol and the Small Sample Protocol 59

62 Table 1. Array quality Figure 2. Image File of Human Focus Array. The array was hybridized by fragmented crna that was amplified from 100 ng of Sample F using the small sample RNA amplification protocol. Results and Discussion A B Table 2. Standard protocol small sample protocol GeneChip hybridization signal intensity. sample 5 standard protocol 1 small sample. C D Figure 3. Scatter Plot for the Comparison between the Small Sample Protocol and the Standard Protocol 60

63 A B Figure 4. Comparison of two different samples prepared by the Standard Protocol (A) and the Small Sample Protocol (B). Conclusion BIOTIMES Table 3. sample standard protocol small sample call concordance analysis (NC: No Change, I: Increase, D: Decrease). References 61

64 Expression microarray reproducibility is improved by optimising purification steps in RNA amplification and labelling By Ali Naderi, Ahmed A Ahmed, Nuno L Barbosa-Morais, Samuel Aparicio, James D Brenton and Carlos Caldas Introduction Results and Discussion Effect of genomic DNA carry over in RNA amplification 62

65 cdna purification affects transcript representation Guanidinium-phenol extraction is the optimal method to purify arna The best method for arna recovery is dependent on total RNA qulaity BIOTIMES 63

66 Removal of protein impurities is essential for arna labeling reaction LiCl is the optimal method for purification of labeled arna 64

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