BIOGNOSTIK. RNAi Catalog How to meet current demands on RNAi experiments?

Transcription

1 BIOGNOSTIK RNAi Catalog 2006 How to meet current demands on RNAi experiments? New publication guidelines for RNAi data: Various controls requested for RNAi experiments Nature Cell Biology 2003 (1) Blast search engine inappropriate for short oligos (2) Up to 75% of commonly used sirnas risk off-target activity (2) Many targets restrictive to RNAi: Secondary structure of the target mrna seen as crucial factor (3, 4)

2 BIOGNOSTIK Experts for Highly Effective Protein Knockdown since 1991 Take advantage of Biognostik s RNAi Solution 1. We design a new generation of highly effective sirnas Proprietary design algorithm based on long-term experience with gene expression inhibitors Multi-step parallel processing analyses the secondary structure Binding affinities of the target mrna sequence are determined In house programs, developed and refined over years, are used for stringent homology checks Latest research results like different end stabilities are included 2. We provide a new concept of cross validation of your results Double checkup with Antisense circumvents cost and time intensive controls Rescue and gene expression controls can be avoided Validated results accepted by referees of scientific journals More than 270 references and several clinical trials document our knowledge regarding highly effective protein knockdown

3 Biognostik s sirnai Design 4 -Excellent design including latest research results and proprietary algorithm -Consideration of different end-stabilities -High specificity - only the antisense strand is taken up by RISC -Target accessibility and binding affinities are evaluated -Determination of sirna internal stability profiles -In house programs for stringent homology checks Table of Contents 3 Antisense & RNAi Kit 6 -Two powerful tools for receiving validated results -Opens the gate for successful publications and drug development -Avoids time intensive and expensive control experiments Custom Designed RNAi Kit 8 -Highly effective sirnas designed for your target gene -Guaranteed silencing at minimum costs Custom Designed Single sirna Probes 9 -Single sirna probe designed for your target gene Custom sirna Synthesis Service 9 -Premium quality synthesis and purification of your provided sequence -Rigorous quality control -Your choice of purification method, amount and format RNAi Controls 10 -An overview about current standards -Biognostik s controls Biognostik s Services 13 -sirna delivery -Assays to detect RNAi effects Technique Description 14 -RNAi mechanism -Antisense technique -A survey References 17 Order Form 18

4 Biognostik s sirna Design 4 -Excellent design including latest research results and proprietary algorithm -Consideration of different end-stabilities -High specificity: only antisense strand is taken up by RISC -Target accessibility and binding affinities are evaluated -Determination of sirna internal stability profiles -In house programs for stringent homology checks Initial work by Tuschl and colleagues resulted in the development of a basic set of guidelines for the selection of sirnas (5, 6). These initial studies focused on the structural properties of sirna, i.e. duplex length and composition of the 3 dinucleotide overhangs. Ongoing research with RNAi has shown that the basic guidelines have to be supplemented with various design criteria like different endstabilities to improve chances that an sirna will be specific (7, 8, 9). Most recent studies have revealed that the secondary structure of the mrna target is of great importance for sirna efficiency (10, 11, 12). This determinant makes even restrictive targets accessible for RNAi. Biognostik s design algorithm includes the key aspects for effective RNAi: 1. GC-content GC-rich target sites should be avoided because of their potential to be involved in strong secondary structures. 2. RISC can use either the sense or antisense strand to guide mrna recognition Sense and antisense strand of an sirna are not equally likely to be bound by the RISC complex. Each RISC in a cell uses only one strand of an sirna as a guide for RNAi, thus the strand that is bound by RISC dictates what mrna sequences are targeted for degradation. Strand selection can be affected by the nucleotide composition of the sirna. The consideration of different end-stabilities is therefore a key item of the design algorithm for: a) Maximizing silencing efficacy since the correct strand is taken up by the RISC complex b) Higher specificity since the sense strand cannot guide the degradation of non-target genes 3. Target accessibility and binding affinities Unlike first assumptions recent publications have shown that the secondary structure of the mrna target is of great importance for sirna efficiency. The folding pattern of the target site as well as the number of bound bases strongly contributes to the potency of gene silencing by an sirna. Biognostik has developed a proprietary computer-aided sequence design and selection technology, which analyses the entire mrna for highly accessible target sites. Biognostik has become an mrna secondary structure expert, because of its long lasting Antisense research.

5 Biognostik s sirna Design 5 to continue: For more than a decade we have constantly collected data and refined the process of elucidating the inherent local mrna structure. Biognostik s multistep analysis of mrna includes e.g.: -Subalgorithms, which are used for calculating the energy of intramolecular nucleotide binding -Analysis of structure characteristics like size, conformation and energy profile 4. sirna internal stability profiles The sirna sequences are examined for intra- and intermolecular binding affinities. Complementary strands with internal repeats favor stable hairpin structures thus exhausting the effective concentration of the functional duplexes. 5. Stringent homology checks Stringent homology checks are an indispensable part of the sirna design process. Even transcripts with only partial identity could be knocked down by a particular sirna (13, 14). Biognostik s RNAi design includes an advanced homology search step. All sequences, which show relevant cross homologies to other GenBank sequences, are excluded. In house programs, which were developed over years to guarantee highly specific sequences are used for identifying and evaluating all possible significant similarities of the sirnas sequences to other genes. Both sirna strands are subjected to customized alignment searches to minimize risks of generating off-target effects. Be aware that the standard Blast search engines are inappropriate for short oligos (2). 6) Additional determinants of the design algorithm -Decreased thermodynamic stabilities in specific regions of the sirna -Distance of the sirna from the start codon -Nucleotide content of the 3 overhangs -Sequences are analyzed for unwanted or toxic sequence motifs -Avoiding of SNPs (if information available) -Base preferences at specific positions Biognostik offers with its comprehensive RNAi design algorithm the solution for effective gene silencing. Researchers avoid testing multiple sirnas to identify potent targets and therefore save time and money. Furthermore highly functional sirnas induce maximal silencing at lower effective concentrations. Lower effective sirnas concentrations are desirable, because the possibility of dose-dependent off-target effects and cellular toxicity is minimized. Induction of stress and apoptosis genes at higher concentrations of sirna was reported in several investigations (13, 14, 15).

6 Antisense & RNAi Kit 6 -Two powerful tools for receiving validated results -Opens the gate for successful publications and drug development -Avoids time and cost intensive control experiments Although the apparent ease of RNAi is striking, it has also led to the application of many papers that display RNAi data without sufficient controls. The problem has become apparent to such a high extent that referees of scientific journals request the issue of standards in the field. They demand complex control experiments or independent other techniques to verify the RNAi results (1). To solve this problem Biognostik has developed an Antisense & RNAi Kit, which provides the researcher a very simple way to fulfill the new requirements. This new concept is based on a cross-validation: we design Antisense and sirna oligonucleotides for the same target mrna. Since Antisense and sirna molecules work through independent mechanisms of action to inhibit target gene expression, a same observed phenotype would verify that the achieved effects are due to a real and specific Antisense and RNAi mechanism. The researcher obtains a double checkup of the experimental results with two different methods. The double check-up can replace cost and time intensive control experiments. No need of multiplicity controls: A cross-validation with two different protein knockout techniques is sufficient to confirm that a phenotype is specific to the reduction of a specific gene expression and not due to off-target effects of a single sirna. Editors of journals accept the cross-validation as one of the best ways to improve confidence in RNAi data and control for sequencespecific off-target effects. No need of global gene expression analysis: You simply can compare the global gene expression pattern of your RNAi results with the Antisense outcome, because Antisense does not induce an interferon response. No need of rescue experiments: sirna were shown to inhibit translation of partially complementary target sequences in a microrna (mirna) like manner. Recently it has been argued that most sirna sequences published to date are partially complementary to several human genes and might cause unintended gene silencing. (2, 16, 17) Antisense cannot mediate an effect like an mirna. Consequently a same observed phenotype with both approaches would verify that the achieved effects are due to a real and specific RNAi mechanism. No loss of time when switching from cell culture to in vivo: Because in vivo there is no need for transfection of Antisense, you can easily switch to the animal model.

7 Antisense & RNAi Kit 7 -We design Antisense and RNAi probes with our proprietary design technology -You get a double check-up of your experimental results with two different methods -Cross validation completely satisfies the demands of referees of scientific journals -Validated results are the first step for successful drug development The Antisense & RNAi Kit includes: -2 highly effective and specific sirna probes designed for your target gene -2 highly effective and specific Antisense probes designed for the same target gene -Guaranteed silencing when used with appropriate controls -sirna and Antisense for every gene listed in GenBank database -High quality synthesis (see next page) -Purified and ready to use probes -Professional technical customer support to assist you in setting up successful experiments in a minimum of time Both methods should be applied separately from each other. We recommend analyzing both mrna and protein levels of your target gene. Antisense & RNAi Kit: Effective and Reliable Protein Knock-down Kit Kit Size Order Code Price Price* Antisense & RNAi Kit 120 2x 100 nmol AS, 2x 20 nmol sirna ,- EURO US$ 1420,- Antisense & RNAi Kit 240 2x 200 nmol AS, 2x 40 nmol sirna ,- EURO US$ 2140,- *only US and Canada

8 Custom Designed RNAi Kit 8 -Highly effective sirnas designed for your target gene -Guaranteed silencing at minimum costs The selection of efficient sirnas relies mainly on the analysis of sequence elements that mediate efficient incorporation into the RISC complex and on mrna target accessibility. Biognostik is the premier supplier who includes the main determinants for highly effective and specific RNAi into its proprietary design algorithm (for a detailed description see page 4). Custom Designed RNAi Kit contains: -3 highly effective and specific sirna probes designed for your target gene -sirna for every gene listed in GenBank database -Full sequence information -Guaranteed silencing* -High quality synthesis** -Purified, annealed and ready to use probes -Professional technical customer support to assist you in setting up successful experiments in a minimum of time *Guaranteed silencing Biognostik guarantees that two of the sirnas will silence target mrna levels by 70% or more when transfected at 100 nm under appropriate transfection conditions. Optimum transfection efficiency must be confirmed using one of Biognostik's positive control sirna or another previously validated functional sirna. Low transfection efficiency will result in lower level of silencing. Transfection efficiency may vary between different cell lines, and should be optimized for each individual cell line. **High quality synthesis Each sirna is synthesized and purified to meet the highest quality standards. As part of a rigorous quality control procedure, the mass of each RNA oligonucleotide is analyzed by MALDI-TOF mass spectrometry. Each annealed sirna is also analyzed by gel electrophoresis to confirm that the strands annealed properly. The result is premium quality sirna that is purified and ready to use. Kit Kit Size Order Code Price Price* CD RNAi Kit 20 3x 20 nmol sirna ,- EURO US$ 940,- CD RNAi Kit 40 3x 40 nmol sirna ,- EURO US$ 1090,- *only US and Canada

9 Single sirna Probes 9 Custom Designed sirna Probes -Single sirna probes designed for your target gene Biognostik offers custom designed single sirna Probes: -1 highly effective and specific sirna probe designed for your target gene -sirna for every gene listed in GenBank database -Full sequence information -High quality synthesis (see Custom Designed sirna Kit on page 8) -Purified, annealed and ready to use probe -Professional technical customer support to assist you in setting up successful experiments in a minimum of time Product Size Order Code Price Price* CD sirna Probe 20 1x 20 nmol sirna ,- EURO US$ 329,- CD sirna Probe 40 1x 40 nmol sirna ,- EURO US$ 379,- *only US and Canada Custom sirna Synthesis Service -Premium quality synthesis and purification of your provided sequence -Rigorous quality control -Your choice of purification method, amount and format Biognostik s Custom sirna Synthesis service provides purified and annealed sirna for effective gene silencing. Simply order your sirna sequence of choice by providing either the 19 base target sequence, or by providing the sense and antisense sirna sequence. For your convenience, sirnas are provided in various formats and amounts. Specific modifications are delivered on request. Each sirna is synthesized and purified to meet the highest quality standards. The standard purification procedure includes deprotection and column purification, and typical yields sirna that is 90% pure (guaranteed > 80% pure). As part of a rigorous quality control procedure, the mass of each RNA oligonucleotide is analyzed by MALDI-TOF mass spectrometry. Each annealed sirna is also analyzed by gel electrophoresis to confirm that the strands annealed properly. The result is premium quality sirna that is purified and ready to use. Product Size Order Code Price Price* sirna Probe 20** 1x 20 nmol sirna ,- EURO US$ 235,- sirna Probe 40** 1x 40 nmol sirna ,- EURO US$ 299,- *only US and Canada **standard purity, annealed, 3 dtdt overhangs; for further formats and modifications, please inquire

10 RNAi Controls 10 An overview about current standards Leaders in the field have recommended a standard set of RNAi controls. The goal is to establish common guidelines for publications regarding interpreting and comparing the silencing results across the growing number of RNAi studies (1). 1. Cells and transfection reagent only These two trials should always serve as baseline experiments. 2. Scrambled sirna control as negative control A negative control should have none or minimal homology with any known target in the cell. This control serves to rule out expression changes due to the introduction of any sirna; substance related effects can be detected. Biognostik s negative sirna control are validated for use in human, mouse and rat cell lines. 3. Positive sirna control A positive control is used to establish optimal transfection conditions and to check the RNAi machinery in a specific cell line. Useful silencing controls include high efficiency sirnas directed against known target genes. GAPDH sirna, for example, is a commonly used positive control because GAPDH is expressed at easily detectable levels in most cell types. 4. Multiplicity controls It is extremely important to use several different specific sirnas per target gene in individual transfections to confirm that a phenotype was specific to the reduction of gene expression and not due to off-target effects of a single sirna. A good way to enhance confidence in RNAi data is to demonstrate a similar effect with two or more sirnas targeted to different sites in the message under study. Alternatively, the RNAi approach is useful supplemented by alternative methods, like Antisense; see also Antisense & RNAi Kit on page 6. Different sirnas to the same target and with comparable gene silencing efficacy should induce similar changes in gene expression profiles or phenotypes. Any changes induced by one sirna and not the other(s) may be attributed to off-target effects. Therefore Biognostik s Custom Design sirna Kit comprises three highly effective and specific sirna probes designed for your target gene, which assists you to control sequence-specific off-target effects. 5. Quantitative control To minimize non-specific effects and prevent oversaturation of the RNAi machinery, researchers are strongly encouraged determining the lowest sirna concentration necessary to produce the desired level of silencing. Decreased levels of sirna minimize the possibility of dose-dependent off-target effects and cellular toxicity (13, 14, 15). To ensure target specificity use highly effective sirnas provided by Biognostik, which often can be applied at concentration < 30 nm.

11 RNAi Controls 11 to continue: 6. Monitor both target mrna and protein level sirna were shown to inhibit translation of partially complementary target sequences in a microrna like manner (16). Thus, it is important to show reduction of expression at the mrna and protein level. If both, mrna and protein level are reduced, the response is classical RNAi. Protein decline in the absence of mrna reduction, however, may indicate that an sirna is mediating its effects at the translation level like a microrna. 7. Analysis of global gene expression and antiviral response Recent investigations reported the induction of the interferon response by short hairpin RNAs or sirnas (18, 19), which can lead to an unintentional activation of global translational repression. Global gene expression can be analysed as a control for any non-specific effects. The most comprehensive way to monitor changes in gene expression patterns or upregulation of the antiviral response is using DNA microarrays. However this may be expensive or impractical in many cases. To avoid time consuming global gene expression studies you can alternatively compare your RNAi results with a parallel Antisense approach. Antisense oligonucleotides do not induce an antiviral response. A supplementation with an alternative protein knockdown technique gives you an equal indication of specific versus non-specific sirna effects. 8. Rescue experiments A rescue control can give the final confirmation that an observed effect is specifically due to knocking down the target of interest. In rescue experiments the RNAi effect is reversed by expressing the target gene in a form that is refractory to silencing by a particular sirna. This is usually accomplished by introduction one or more silent base pair changes into the target gene at the sirna target site. Such an sirna-resistant form of a gene can be cloned into expression plasmids and introduced into the cell via transfection. Because of a silent mutation in the 3 nucleotide of a codon, the transcript from the refractory target gene encodes a functional protein that is insensitive to sirna-induced silencing. Thus, the wild type should be restored in cells co-transfected with the sirna and the plasmid encoding the refractory target. We open the way for your RNAi success. A recent editorial in Nature Cell Biology terms the rescue control as ultimate control for sirna experiments. Unfortunately these experiments are time consuming and expensive. As an alternative researcher have the opportunity to supplement the RNAi experiments with different methods, like Antisense (1). For that Biognostik offers Antisense & RNAi Kits, which avoid time and cost extensive control steps. Using Antisense and RNAi in parallel offers a powerful tool to get validated results for publications and to speed up the development of new drugs.

12 Biognostik s Controls 12 -Positive controls ideal for optimizing sirna experimental conditions -Negative controls validated for use in human, mouse and rat cell lines -Purified, duplexed and ready to use Positive Controls (5 nmol + 2 nmol Negative Control) A premade gene specific positive control is used to establish optimal transfection conditions and to check the RNAi machinery in a specific cell line. Negative Controls (5 nmol) These negative controls have no significant homology with human, mouse or rat gene sequences. They serve to rule out unspecific effects on gene expression. Positive Control Size Order Code Price Price* GAPDH 5 nmol (incl. 2 nmol neg. control) x ,- EURO US$ 255,- beta-actin 5 nmol (incl. 2 nmol neg. control) x ,- EURO US$ 255,- *only US and Canada Negative Control Size Order Code Price Price* Neg. Control 1 5 nmol negative control ,- EURO US$ 205,- Neg. Control 2 5 nmol negative control ,- EURO US$ 205,- *only US and Canada

13 Biognostik s Services 13 -We assist you to get through the jungle of different methods -Detailed literature research -Comparison of different methods -Protocols for your cell culture sirna delivery For transfection or electroporation methods we advice customers regarding their experimental setups and name corresponding providers. This includes detailed literature research and comparison of different methods and protocols. Assays to detect RNAi effects Gene silencing can be detected by 1) Measuring the changes on the level of the target mrna 2) Analyzing the protein quantity 3) Functional assays We recommend analyzing both mrna and protein levels. Since sirnas exert their effects at the mrna level the first assay for sirna validation is one that monitors target mrna levels. The most popular and commonly used technique for quantifying mrna levels is qrt-pcr. An accompanying method for evaluation sirna effects is monitoring the protein level. The most preferred techniques are Western Blot, ELISA or Immunoprecipitation. In addition to assays that quantify the effects of sirna on mrna and protein levels, functional assays designed to examine one or more phenotypes affected by the target protein can be performed. Summary For performing successful RNAi experiments in mammalian cell systems, you need a target specific sirna that silences your gene of interest, proper controls, an sirna delivery method and an assay to detect the RNAi effect. Biognostik provides custom designed sirna probes (in kit format or as single probes) and a set of proper controls. Furthermore we offer an Antisense & RNAi Kit, a so far unique technique of highly specific protein knockdown, which offers a reliable method to validate your results. For sirna delivery and assays to detect the RNAi effect we assist the customer to find the right supplier.

14 RNAi Mechanism 14 RNAi interference (RNAi) is the mechanism of sequence-specific, posttranscriptional gene silencing initiated by double-stranded RNAs (dsrna) homologous to the gene being suppressed. RNAi represents an ancient and evolutionarily conserved system considered to protect the genome against viral infections and genomic instability caused by mobile genetic elements such as transposons, which produce dsrna intermediates inside the cell. In most eukaryotic cells, RNAi occurs by the cleavage of long doublestranded RNA into nucleotide short-interfering RNA (sirna). These sirnas become part of an intracellular RNA-induced silencing complex (RISC). RISC separates the sirnas into single strands and scans for complementary cellular mrna. Upon hybridization the target mrna is cleaved by RISC, which leads to specific gene silencing. In mammalian systems RNAi is introduced by the introduction of sirna, since long dsrna induces a nonspecific interferon response. The application of RNAi in mammals offers a powerful process to regulate gene expression to determine the role of specific proteins in biological processes. Besides gene function analysis RNAi is an elegant tool to speed up the drug target validation process. RNAi mediated downregulation of a receptor or enzyme can quickly answer the question of whether the development of a small-molecular antagonist to this protein will have the anticipated biological and therapeutic effects.

15 Antisense Technology 15 Antisense oligonucleotides are designed to hybridize to their specific mrna target. This hybrid formation causes a steric or conformational obstacle for protein translation. As a result, the production of a specific protein is temporarily inhibited without affecting the expression of other genes and without intervention on the gene level. Biognostik is the world s foremost supplier of custom-designed, high quality phosphorothioate Antisense oligonucleotides that specifically inhibit gene expression in cell lines and in vivo. More than 270 references and several clinical trials document impressively our knowledge in this field. A B A: Normal flow of genetic information, where a gene is transcribed into mrna and translated into the corresponding protein. B: Antisense oligonucleotides are taken up by the cell, hybridize to the target mrna and block protein expression. Antisense oligonucleotide hybridizes to the complementary target mrna and blocks translation of the protein.

16 Antisense & RNAi: A Survey 16 Currently the Antisense and RNAi techniques are the best methods for protein knockdown. Both revolutionize the way researcher study gene function. Using both techniques in parallel offers a powerful process to get validated results for publications and to speed up the development of new drugs. Subject Antisense RNAi Design appropriate design process required appropriate design process required Cell Culture highly effective highly effective In vivo highly effective, no need of transfection no broad established procedure so far Negative Control required required Positive Control not required required Multiplicity Control not required, no mirna effect required* Quantitative Control not required, no transfection step required, transfection control Analysis of Global Gene Expression not required required* Rescue Experiments not required required* Assays protein expression mrna and protein expression *Can be avoided when using the Antisense & RNAi Kit - for further explanation see control chapter on page 10.

17 References 17 (1) "Whither RNAi?", (2003), Nat Cell Biol 5(6): (2) Snove, O., Jr. and T. Holen (2004). "Many commonly used sirnas risk off-target activity." Biochem Biophys Res Commun 319(1): (3) Luo, K. Q. and D. C. Chang (2004). "The gene-silencing efficiency of sirna is strongly dependent on the local structure of mrna at the targeted region." Biochem Biophys Res Commun 318(1): (4) Kretschmer-Kazemi Far, R. and G. Sczakiel (2003). "The activity of sirna in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides." Nucleic Acids Res 31(15): (5) Elbashir, S. M., W. Lendeckel, et al. (2001). "RNA interference is mediated by 21- and 22-nucleotide RNAs." Genes Dev 15(2): (6) Elbashir, S. M., J. Harborth, et al. (2002). "Analysis of gene function in somatic mammalian cells using small interfering RNAs." Methods 26(2): (7) Reynolds, A., D. Leake, et al. (2004). "Rational sirna design for RNA interference." Nat Biotechnol 22(3): (8) Schwarz, D. S., G. Hutvagner, et al. (2003). "Asymmetry in the assembly of the RNAi enzyme complex." Cell 115(2): (9) Khvorova, A., A. Reynolds, et al. (2003). "Functional sirnas and mirnas exhibit strand bias." Cell 115(2): (10) Schubert, S., A. Grunweller, et al. (2005). "Local RNA target structure influences sirna efficacy: systematic analysis of intentionally designed binding regions." J Mol Biol 348(4): (11) Heale, B. S., H. S. Soifer, et al. (2005). "sirna target site secondary structure predictions using local stable substructures." Nucleic Acids Res 33(3): e30. (12) Yiu, S. M., P. W. Wong, et al. (2005). "Filtering of ineffective sirnas and improved sirna design tool." Bioinformatics 21(2): (13) Jackson, A. L., S. R. Bartz, et al. (2003). "Expression profiling reveals off-target gene regulation by RNAi." Nat Biotechnol 21(6): (14) Semizarov, D., L. Frost, et al. (2003). "Specificity of short interfering RNA determined through gene expression signatures." Proc Natl Acad Sci U S A 100(11): (15) Persengiev, S. P., X. Zhu, et al. (2004). "Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (sirnas)." Rna 10(1): (16) Doench, J. G., C. P. Petersen, et al. (2003). "sirnas can function as mirnas." Genes Dev 17(4): (17) Scacheri, P. C., O. Rozenblatt-Rosen, et al. (2004). "Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells." Proc Natl Acad Sci U S A 101(7): (18) Bridge, A. J., S. Pebernard, et al. (2003). "Induction of an interferon response by RNAi vectors in mammalian cells." Nat Genet 34(3): (19) Sledz, C. A., M. Holko, et al. (2003). "Activation of the interferon system by short-interfering RNAs." Nat Cell Biol 5(9):

18 Order Form Part 1/2 18 Ship to Name Department Institution/company Address (no PO Box) City, post code Phone Fax (very important) Bill to Name Department Institution/company Abroad: VAT ID for countries of the E.C. Address (no PO Box) City, post code Phone Fax (very important) Your internal order No. Please type clearly. 1. Please indicate product type and size. For detailed description see corresponding catalog pages. ANTISENSE & RNAi Kit (page 6-7) Custom Designed RNAi Kit (page 8) Custom Designed Single sirna Probes (page 9) Antisense & RNAi Kit 120 Order Code: CD RNAi Kit 20 Order Code: CD sirna Probe 20 Order Code: Antisense & RNAi Kit 240 Order Code: CD RNAi Kit 40 Order Code: CD sirna Probe 40 Order Code: For the design process, please provide name and species of your target gene and the corresponding GenBank Accession Number of the mrna or cdna. You will find the Accession No. at If you need assistance, please contact us. GenBank / EMBL Accession No.* Name of Gene (*cdna or mrna only) Species Please check your order for completeness to avoid delay in processing. An order confirmation will be sent by fax soon after receiving the order. By ordering I accept Biognostik s "Ordering & Terms" as stated on our webpage: Date Signature Please copy and FAX back to: + 49-(0)

19 Order Form Part 2/2 19 Ship to Name Department Institution/company Address (no PO Box) City, post code Phone Fax (very important) Bill to Name Department Institution/company Abroad: VAT ID for countries of the E.C. Address (no PO Box) City, post code Phone Fax (very important) Your internal order No. Custom sirna Synthesis Service (page 9) You can order your sirna sequence of choice by providing either the 19 base target sequence, or by providing the sense and antisense sirna sequence via to 1. Please indicate product type and size *standard purity, annealed, 3 dtdt overhangs; for further formats and modifications, please inquire 5 sirna Probe 20* Order Code: sirna Probe 40* Order Code: Please enter the target sequence of the ODN below, from 5 to 3 and by using g instead of G ; Please type clearly to avoid a mix-up. 3 Positive Controls (incl. 2 nmol neg. control) (page 12) GAPDH Order Code: x beta-actin Order Code: x Please indicate species Negative Controls (page 12) Neg. Control 1 Order Code: Neg. Control 2 Order Code: Please check your order for completeness to avoid delay in processing. An order confirmation will be sent by fax soon after receiving the order. By ordering I accept Biognostik s "Ordering & Terms" as stated on our webpage: Date Signature Please copy and FAX back to: + 49-(0)

20 Copyright and Trademarks No part of this catalog may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the written permission of the publisher. For information regarding permission please contact Biognostik GmbH. and ANTISENSE are registered trademarks of Biognostik GmbH, Göttingen, Germany. 20