Silencer sirna Cocktail Kit (RNase III) (Cat #1625) Instruction Manual

Transcription

1 Silencer sirna Cocktail Kit (RNase III) (Cat #1625) Instruction Manual I. Introduction A. Background B. Reagents Provided with the Kit and Storage C. Materials Not Provided with the Kit D. Related Products Available from Ambion II. Preparation of Template DNA A. Choosing the Target Site B. Strategies for Transcription of dsrna C. PCR Templates D. Plasmid Templates III. Silencer sirna Cocktail Kit Protocol A. Before Using the Kit for the First Time B. Transcription Reaction Assembly C. Annealing RNA to Maximize Duplex Yield D. Nuclease Digestion to Remove DNA and ssrna E. Purification of dsrna F. RNase III Digestion and sirna Purification G. sirna Cocktail Quantification H. Transfecting Mammalian Cells IV. Troubleshooting A. Use of the GAPDH Control Template B. Troubleshooting Low Yield from the Transcription Reaction C. Troubleshooting Unexpected Transcription Reaction Products D. Troubleshooting RNase III Digestion and sirna Purification V. Additional Procedures A. Quantitation of RNA by Spectrophotometry B. Agarose and Acrylamide Gel Electrophoresis Instructions VI. Appendix A. References B. Silencer sirna Cocktail Kit Specifications C. Aqueous Solution Material Safety Data Sheet

2 Manual Version 0305 Literature Citation When describing a procedure for publication using this product, we would appreciate that you refer to it as the Silencer sirna Cocktail Kit (RNase III). If a paper that cites one of Ambion s products is published in a research journal, the author(s) may receive a free Ambion T-shirt by sending in the completed form at the back of this instruction manual, along with a copy of the paper. Licensing Statement Ambion has been granted rights by the Massachusetts Institute of Technology to US Patent Applications 60/265232, 09/ and PCT/US01/10188, RNA Sequence- Specific Mediators of RNA Interference. This product is covered by several patent applications owned by STANFORD. The purchase of this product conveys the buyer the limited, non-exclusive, non-transferable right (without the right to resell, repackage, or further sublicense) under these patent rights to perform the sirna production methods claimed in those patent applications for research purposes solely in conjunction with this product. No other license is granted to the buyer whether expressly, by implication, by estoppel or otherwise. In particular, the purchase of this product does not include nor carry any right or license to use, develop, otherwise exploit this product commercially, and no rights are conveyed to the buyer to use the product or components of the product for any other purposes, including without limitation, provision of services to a third party, generation of commercial databases, or clinical diagnostics or therapeutics. In addition, any user that purchases more that $5,000 in any calendar quarter may be outside the above research license and will contact STANFORD for a license. This product is sold pursuant to a license from STANFORD, and STANFORD reserves all other rights under these patent rights. For information on purchasing a license to the patent rights for uses other than in conjunction with this product or to use this product for purposes other than research, please contact STANFORD at, This is STANFORD reference S Warranty and Liability Ambion is committed to providing the highest quality reagents at competitive prices. Ambion warrants that the products meet or exceed the performance standards described in the product specification sheets. If you are not completely satisfied with any product, our policy is to replace the product or credit the full purchase price and delivery charge. No other warranties of any kind, expressed or implied, are provided by Ambion. Ambion s liability shall not exceed the purchase price of the product. Ambion shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products. This product is intended for research use only. This product is not intended for diagnostic or drug purposes. This product is covered by US patent 5,256,555 and US patents pending. SUPERase In is covered by US and foreign patents pending. Copyright pending by Ambion, Inc. all rights reserved.

3 Introduction I. Introduction A. Background The Silencer sirna Cocktail Kit (RNase III) (patents pending) is a system for in vitro transcription of long dsrna and its enzymatic conversion to sirna for use in RNAi in mammalian cells. Template DNA provided by the user is transcribed using Ambion s powerful MEGAscript large scale transcription technology to synthesize dsrna. The dsrna is digested by RNase III into a mixture or cocktail of sirna molecules capable of inducing the RNAi effect in mammalian cells in a very specific fashion (Byrom et al. 2003). Using this methodology, a large region of mrna can be targeted for RNAi with a single reaction. RNA interference (RNAi) and short interfering RNAs (sirnas) bp sirnas?? RISC Cell Membrane RNase sirnas associate with RISC complex Cleavage of mrna by RISC? long dsrna dsrna cleavage Degradation by exonucleases RNAi, the phenomenon by which dsrna specifically suppress expression of a target gene, was originally discovered in worms (Fire et al. 1998), but has now been found in a large number of organisms, including flies (Misquitta and Paterson 1999), trypanosomes (Ngo et al. 1998), planaria (Sánchez-Alvarado and Newmark 1999), hydra (Lohmann et al. 1999), and zebrafish (Wargelius et al. 1999). RNAi is becoming one of the most widely used methods for studying gene function in mammalian cells. In the RNA interference (RNAi) pathway in vivo, long double-stranded RNA (dsrna) introduced into a cell is cleaved into a mixture of short dsrna molecules called short interfering RNA (sirna). The enzyme that catalyzes the cleavage is called Dicer; it is an endo-rnase that contains RNase III domains. The sirnas produced by Dicer are bp in length and contain 3' dinucleotide overhangs with 5'-phosphate and 3'-hydroxyl termini (Bernstein et al. 2001, Elbashir et al. 2001, Grishok et al. 2001, Hamilton and Baulcombe 1999, Knight and Bass 2001, Zamore et al. 2000). sirnas associate with a cellular complex called the RNA-induced silencing complex (RISC) and guides the complex to their target mrna through base pairing interactions (Hammond et al. 2001). Once the RISC associates with the target mrna, nucleases cleave the mrna (Tuschl et al. 1999, Zamore et al. 2000), causing a reduction in target gene expression. RNA Interference in vivo I.A. Background 1

4 Silencer sirna Cocktail Kit (RNase III) Silencer sirna Cocktail Kit procedure overview Transcription 2 4 hr Nuclease Digestion (37 C) 1 hr dsrna Purification min RNase III Digestion (37 C) 1 hr sirna Purification 10 min The Silencer sirna Cocktail Kit procedure mimics the process of sirna production in vivo. It begins with a high yield transcription reaction to synthesize two complementary RNA transcripts from templates supplied by the user. Next, the transcription template(s) and any single-stranded RNA (ssrna) are removed by nuclease digestion, and the long dsrna is purified with a solid-phase adsorption system (Transcription Reaction Filter Cartridges). The long dsrna is then cleaved with RNase III in a 1 hour reaction to produce sirna. The recombinant E. coli RNase III used for this reaction cleaves long dsrna into short bp dsr- NAs containing a 5'-PO 4, a 3'-OH, and a 2 nucleotide 3' overhang; the same structure as sirna produced in vivo. Finally the sirna is purified using the included sirna Purification Units to remove any undigested or partially digested material. After purification, the sirna can be used for inducing RNAi by introduction into mammalian cells. We and others have demonstrated that sirnas generated using bacterial RNase III are capable of efficiently and specifically inducing RNAi in mammalian cells (Yang et al. 2002, Calegari et al. 2002, Byrom et al. 2003, Trotta et al. in press). 5' 3' 5' 3' 5' 3' Transfection Advantages of the Silencer sirna Cocktail Kit approach to making sirna Methods for producing sirna include chemical synthesis, in vitro transcription (e.g. using this kit the Silencer sirna Cocktail Kit or Ambion s Silencer sirna Construction Kit), and using sirna expression vectors (e.g. Ambion s psilencer vectors). The Silencer sirna Cocktail Kit approach eliminates the need to screen several different sirna to identify an active one, making it both convenient and economical. Since the sirnas are generated from long dsrna precursors, they contain a mixture of sirna target sites. Generally, we observe that one out of four individual sirnas can reduce the expression of the target by 80% or more. Thus, synthesizing a 150 bp dsrna, and RNase III digesting it into an sirna cocktail using this kit, the chance that the mixture will contain a functional sirna approaches 100%. Using the Silencer sirna Cocktail Kit minimizes not only the cost of making sirna but also the time required for performing RNAi experiments in mammalian cells. The amount of sirna generated per transcription reaction is enough for hundreds of knockdown experiments in 24 well tissue culture plates. 2 I.A. Background

5 Introduction B. Reagents Provided with the Kit and Storage The kit contains components to synthesize 20 dsrnas, and to digest a total of 600 µg of dsrna into sirna cocktails. Properly stored kits are guaranteed for 6 months from the date received. Amount Component Storage 20 Transcription Rxn Filter Cartridges room temp 60 Collection Tubes room temp 20 sirna Purification Units room temp 40 µl T7 Enzyme Mix 20 C T7 RNA polymerase, SUPERase In, and other components in buffered 50% glycerol 40 µl 10X T7 Reaction Buffer 20 C 40 µl ATP Solution (75 mm) 20 C 40 µl CTP Solution (75 mm) 20 C 40 µl GTP Solution (75 mm) 20 C 40 µl UTP Solution (75 mm) 20 C 1ml 10X Binding Buffer 20 C 10 ml Nuclease-free Water 20 C 4 ml Elution Solution 20 C 40 µl RNase A 20 C 600 µl RNase III (1U/µl) 20 C 500 µl 10X RNase III Buffer 20 C 45 µl DNase I 20 C 100 µl 10X Digestion Buffer 20 C 12 ml 2X Wash Solution 20 C Add 12 ml 100% ethanol before use 10 µl GAPDH Control Template (100 ng/µl) 20 C I.B. Reagents Provided with the Kit and Storage 3

6 Silencer sirna Cocktail Kit (RNase III) C. Materials Not Provided with the Kit Gene-specific template(s) A transcription template(s) is needed with T7 RNA polymerase promoters positioned to transcribe sense and antisense RNA corresponding to the target RNA. See section II starting on page 6 for a detailed discussion of template requirements and preparation. For dsrna purification: 100% ethanol: ACS grade or better Equipment to draw solutions through the Transcription Reaction Filter Cartridges: use either a microcentrifuge capable of at least 8,000 X g, or a vacuum manifold with sterile 5 ml syringe barrels mounted to support the Transcription Reaction Filter Cartridges. To assess the reaction products: Reagents and equipment for acrylamide gel electrophoresis Spectrophotometer D. Related Products Available from Ambion Silencer Transfection Kit Cat #1630 RNase III Cat #2290 sirna Purification Units Cat #10074G Silencer sirna Labeling Kit Cat #1632, 1634 The Silencer sirna Transfection Kit contains both siport Amine and siport Lipid Transfection Agents and a well-characterized GAPDH sirna, ideal for developing an optimal transfection protocol for your cells. Also included are the corresponding GAPDH negative control sirna, and a detailed instruction manual. Escherichia coli Ribonuclease III (RNase III; EC ) is a double-stranded RNA (dsrna) specific endoribonuclease. It cleaves dsrna into bp dsrna fragments with 2 3 nucleotide 3' overhangs, and 5'-phosphate and 3'-hydroxyl termini. The termini and overhangs of RNase III cleavage products are thus the same as those produced by Dicer in the eukaryotic RNAi pathway. Transfection of RNase III cleavage products can be used to induce RNAi in mammalian cells (patent pending). These are the same sirna Purification Units as those used in the Silencer sirna Cocktail Kit; they have a molecular weight cut-off of 30 kda. They can be used to separate sirna prepared by enzymatic digestion from its longer dsrna substrate. The Silencer sirna Labeling Kits are used for labeling sirna synthesized with the Silencer sirna Construction Kit or synthesized chemically. Labeled sirna can be used to analyze the subcellular distribution of sirna, in vivo stability, transfection efficiency, or the capability of the sirna to attenuate target gene expression. 4 I.C. Materials Not Provided with the Kit

7 Introduction Silencer sirna Controls Cat # Negative Control sirna see Catalog or Web site RNaseZap Cat # Electrophoresis Reagents see Catalog or Web site The Silencer sirna Controls are ready-to-use, chemically synthesized, purified GAPDH, Cyclophilin, or c-myc sirna for up to 150 transfections (of a single well in a 24 well plate). Corresponding scrambled sirna negative controls are also included. These Controls are ideal for developing and optimizing sirna experiments and have been validated for use in mammalian cell lines. Universal scrambled sirna control sequences are available separately as either prepared and tested sirna or templates for use in the Silencer sirna Construction Kit. The scrambled controls have no significant homology to mouse, rat, or human gene sequences and are ideal for use as negative controls in any sirna experiment. RNase Decontamination Solution. RNaseZap is simply sprayed or poured onto surfaces to instantly inactivate RNases. Rinsing twice with distilled water will eliminate all traces of RNase and RNaseZap. Ambion offers gel loading solutions, agaroses, acrylamide solutions, powdered gel buffer mixes, nuclease-free water, and RNA and DNA molecular weight markers for electrophoresis. Please see our catalog or our web site ( for a complete listing as this product line is always growing. I.D. Related Products Available from Ambion 5

8 Silencer sirna Cocktail Kit (RNase III) II. Preparation of Template DNA A. Choosing the Target Site Experiments at Ambion using the Silencer sirna Cocktail Kit have not shown a difference in the effectiveness of sirna cocktails targeting different parts of mrna (i.e 3' end, 5' end or the center of the message). As far as we know, sirna cocktails targeting any part of an mrna can be used for RNAi. In the literature, dsrnas ranging in size from bp have been used as substrates for bacterial RNase III to produce sirna for gene silencing experiments (Yang et al. 2002, Calegari et al. 2002). We find, however, that dsrnas from bp in length provide the highest level of RNAi. With these smaller dsrna substrates, the RNase III digestion yields an sirna cocktail with fewer unique sirnas, but each one is at a higher concentration than when a larger dsrna subtrate is digested. A 200 bp dsrna is typically digested into 7 16 different sirnas. Since generally, one in four sirnas have RNAi activity when introducted into cells, bp dsrnas are extremely likely to yield an sirna cocktail that effectively induces RNAi. B. Strategies for Transcription of dsrna RNase III requires a dsrna template. Since the T7 RNA polymerase used in this kit synthesizes single-stranded RNA (ssrna), use one of the following strategies to produce dsrna: Prepare one DNA template with opposing T7 promoters at the 5' ends of each strand, and use it in a single transcription reaction to synthesize dsrna without a separate annealing step. Use two DNA templates that are identical except that a single T7 promoter sits at opposite ends of the region to be transcribed. With this strategy, the templates are both added to a single reaction. Although both templates should transcribe at the same rate, if they don't, the final dsrna yield will be dictated by the template with the lower transcription efficiency. Alternatively, the two templates can be transcribed in separate reactions to make complementary RNA molecules which are then mixed and annealed. For the annealing step, the two transcripts can be mixed in precisely equimolar amounts. 6 II.A. Choosing the Target Site

9 Preparation of Template DNA Note, however, that if the templates are transcribed in separate transcription reactions, this kit contains enough reagents to produce only 10 different dsrnas. +1 5'-TAATACGACTCACTATAGGGAGA-3' Figure 1. T7 Polymerase Promoter: Minimal Sequence Requirement The +1 base (in bold) is the first base incorporated into RNA. The underline shows the minimum promoter sequence needed for efficient transcription. C. PCR Templates 1. Amplification strategies to add T7 promoter sequences to DNA T7 promoter sequences can be added to DNA using PCR; this generates templates that can be directly added to the Silencer sirna Cocktail Kit transcription reaction. Synthesize PCR primers with the T7 promoter sequence appended to the 5' end of the primer (see Figure 3 on page 8). The T7 promoter-containing PCR primers (sense and antisense) can either be used in separate PCRs, or in a single PCR to generate transcription template for both strands of the dsrna. Figure 2. Adding T7 promoters by PCR Two separate PCRs with a single T7 promoter-containing PCR primer in each A single PCR with the T7 promoter appended to both PCR primers T7 gene specific gene specific T7 gene specific gene specific gene specific T7 gene specific T7 Typically the yield of PCR product is higher with this strategy than if both primers include a T7 promoter. This requires 4 PCR primers and 2 PCRs. After transcription, the RNA products from each reaction will require a separate annealing step to make dsrna. Yield may be lower than if only one primer includes a T7 promoter. Only 2 PCR primers and a single PCR are needed to make template for the dsrna. If the RNA products are 800 nt, an annealing step will not be needed after the transcription reaction; dsrna will form during the transcription reaction. 2. PCR amplification profile suggestions Calculate separately the annealing temperatures of the entire PCR primer (with the T7 promoter site) and the gene specific portion of the PCR primer. II.C. PCR Templates 7

10 Silencer sirna Cocktail Kit (RNase III) Figure 3. Strategy for Adding a Single T7 Promoter by PCR Since the first cycles of PCR use only the 3' half of the PCR primer(s), the gene specific part, the annealing temperature for the first 5 PCR cycles should be ~5 C higher than the calculated T m for the gene-specific region of the primer. We have found that using the calculated annealing temperature often results in synthesis of spurious PCR products. Once some PCR product is made, subsequent primer annealing events can use the entire primer site; therefore use the calculated T m for the entire PCR primer plus ~5 C for cycle 6 and thereafter. We recommend using primers at 100 nm in the PCR mix. Higher concentrations may result in synthesis of primer dimers. T7 PCR primer T7 promoter +1 5'-TAATACGACTCACTATAGGGTACTTGCATTACCCCTCGA-3' Target DNA +1 5'-TGCATTACCCCTCGAATT...GTGATCAGATGCTAGGTAC-3' 3'-ACGTAATGGGGAGCTTAA...CACTAGTCTACGATCCATG-5' PCR 3'-AGTCTACGATCCATG-5' PCR Primer 5'-TAATACGACTCACTATAGGGTACTTGCATTACCCCTCGAATT...GTGATCAGATGCTAGGTAC-3' 3'-ATTATGCTGAGTGATATCCCATGAACGTAATGGGGAGCTTAA...CACTAGTCTACGATCCATG-5' Trxn RNA Transcript 5'-pppGGGUACUUGCAUUACCCCUCGAAUU...GUGAUCAGAUGCUAGGUAC-3' 3. Check PCR products on a gel before using them in this procedure 4. Purification of the PCR products PCR products should be examined on an agarose gel prior to in vitro transcription to estimate concentration and to verify that the products are unique and of the expected size. If the PCR yields multiple bands on a gel, it may be necessary to gel purify the correct size PCR product using standard purification techniques. Note that optimizing the PCR conditions may result in amplification of a single band. 8 II.C. PCR Templates

11 Preparation of Template DNA D. Plasmid Templates 1. Cloning strategy In general, to use plasmid templates in the Silencer sirna Cocktail Kit, it is best to make two separate clones with the same target region in both orientations. Figure 4. Cloning in plasmids T 7 Promoter T 7 Promoter PCR products can be cloned into plasmid vectors using any of the following strategies: Amplify the target by PCR and ligate the product into a PCR vector with a T7 promoter. Identify plasmids with the insert in both orientations with regard to the T7 promoter. Include the T7 promoter sequence to the 5' end of one or both of the PCR primers, then perform PCR to incorporate them into the fragment (see Figure 3 on page 8), finally ligate the PCR product into a PCR cloning vector (that doesn t have a T7 promoter). Include both a T7 promoter and a restriction site at the 5' end of one or both PCR primers to incorporate them into the fragment during PCR, then ligate the PCR product into a cloning vector via the restriction sites. We routinely use all types of restriction enzymes, however, there has been one report of low level transcription from the inappropriate template strand in plasmids cut with restriction enzymes leaving 3' overhanging ends (produced by Kpn I, Pst I, etc.; Schendorn and Mierindorf, 1985). 2. Plasmid linearization Plasmid templates must be linearized downstream of the insert to create a transcription termination site the RNA polymerase will literally fall off the end of the DNA molecule. Linearize each template, then examine the DNA on a gel to confirm that cleavage is complete. Since initiation of transcription is one of the limiting steps of in vitro transcription reactions, even a small amount of circular plasmid in a template prep will generate a large proportion of transcript, wasting much of the synthetic capacity of the reaction. Figure 5. Linearized plasmids T 7 T 7 II.D. Plasmid Templates 9

12 Silencer sirna Cocktail Kit (RNase III) 3. After linearization Terminate the restriction digest by adding the following: 1/20th volume 0.5 M EDTA 1/10th volume of either 3 M NaOAc or 5 M NH 4 OAc 2 volumes of ethanol Mix well and chill at 20 C for at least 15 min. Then pellet the DNA for 15 min in a microcentrifuge at top speed. Remove the supernatant, re-spin the tube for a few seconds, and remove the residual fluid with a very fine-tipped pipet. Resuspend in TE (10 mm Tris-HCl ph 8, 1 mm EDTA) at a concentration of µg/µl. 4. Plasmid DNA purity DNA should be relatively free of contaminating proteins and RNA. We observe the greatest yields of dsrna with very clean template preparations. Most commercially available plasmid preparation systems yield DNA that works well in the Silencer sirna Cocktail Kit. Note that DNA from some miniprep procedures may be contaminated with residual RNase A. Also, restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When transcription from a template is suboptimal, it is often helpful to treat the template DNA with proteinase K ( µg/ml) and 0.5% SDS for 30 min at 50 C, follow this with phenol/chloroform extraction (using an equal volume) and ethanol precipitation. 10 II.D. Plasmid Templates

13 Silencer sirna Cocktail Kit Protocol III. Silencer sirna Cocktail Kit Protocol A. Before Using the Kit for the First Time Prepare the Wash Solution 1. Add 12 ml ACS grade 100% ethanol to the bottle labeled 2X Wash Solution. 2. Mix well, and store at room temperature. We suggest crossing out the 2X from the bottle label after adding the ethanol. In these instructions this reagent will be called Wash Solution once the ethanol is added. B. Transcription Reaction Assembly 1. Thaw the frozen reagents at room temp and place them in ice 2. Assemble transcription reaction at room temperature Remove the T7 Enzyme Mix from the freezer and place it directly in ice; it is stored in glycerol and will not freeze at 20 C. Vortex the 10X T7 Reaction Buffer and the 4 ribonucleotide solutions (ATP, CTP, GTP, and UTP) until they are completely in solution. Once they are thawed, store the ribonucleotides (ATP, CTP, GTP, and UTP) on ice, but keep the 10X Reaction Buffer at room temperature. Microfuge all reagents briefly before opening to prevent loss and/or contamination of any material on the rim of the tube. The reaction should be assembled at room temp because the spermidine in the 10X Transcription Buffer can coprecipitate with the template DNA if the reaction is assembled on ice. Add the 10X Reaction Buffer after the water and template DNA are already in the tube. III.A. Before Using the Kit for the First Time 11

14 Silencer sirna Cocktail Kit (RNase III) The following amounts are for a single 20 µl transcription reaction. Reactions may be scaled up or down if desired. Amount Component EGAscript to 20 µl Nuclease-free Water µg Linear template DNA* 2µl 10X T7 Reaction Buffer 2µl ATP Solution 2µl CTP Solution 2µl GTP Solution 2µl UTP Solution 2µl T7 Enzyme Mix * Templates <500 bp: Use ng of template with T7 promoters flanking the transcription template, or use ng of each template when the T7 promoter template is on separate molecules. Templates 500 bp: Use 1 µg of a template with T7 promoters flanking the transcription template, or use 1 µg of each template when the T7 promoter template is on separate molecules. 3. Mix thoroughly Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube. 4. Incubate at 37 C for 2 hr Incubate the transcription reaction for 2 hr at 37 C. C. Annealing RNA to Maximize Duplex Yield Annealing the complementary RNA is often unnecessary for transcripts 800 nt made from a single template with opposing T7 promoters, because RNA products in this size range will typically hybridize during the transcription reaction. With transcripts >800 nt, however, at least a portion of the transcripts form aggregates (presumably branched structures) that remain in the well of agarose gels during electrophoresis unless the RNA is subjected to a heat denaturation step as described below. Include this annealing step for all >800 nt dsrna synthesis reactions. Include this annealing step for production of 800 nt dsrna when the two strands were synthesized from separate transcription templates (in the same or in separate transcription reactions). 12 III.C. Annealing RNA to Maximize Duplex Yield

15 Silencer sirna Cocktail Kit Protocol 1. Mix the transcription reactions containing complementary RNA 2. Incubate at 75 for 5 min, then cool to room temperature 3. (optional) Check 1/400 th of the dsrna on an agarose gel If sense and antisense RNA was synthesized in a single transcription reaction, both strands of RNA will already be in a single tube, simply proceed to step 2. If desired, reserve a 0.5 µl aliquot of each template for gel analysis. Incubate at 75 C for 5 min then leave the mixture on the bench to cool to room temperature. The RNA will anneal as it cools, forming dsrna. Run 1/400 th of the dsrna on a 1% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation. 1/400 th of a 20 µl dsrna solution is 5 µl of a 1:100 dilution 1/400 th of a 40 µl dsrna solution is 5 µl of a 1:50 dilution Dilute the gel samples in TE (10 mm Tris, 1 mm EDTA) or in gel loading buffer (Instructions for running the gel are in section V.B.2 on page 27.) The dsrna will migrate slightly slower than DNA markers of the same length. D. Nuclease Digestion to Remove DNA and ssrna This DNase/RNase A treatment digests template DNA and any ssrna remaining in the dsrna. Using the reaction conditions specified below, the RNase A will not degrade dsrna. 1. Assemble RNase A digestion reaction on ice 2. Incubate at 37 C for 1 hr The amounts shown are for a 20 µl transcription reaction; scale up if your transcription reaction was larger. Amount Component 20 µl dsrna (from step B.4 or step C.2) 21 µl Nuclease-free Water 5µl 10X Digestion Buffer 2 µl DNase I 2µl RNase A The ssrna will be digested after 15 min but allow the incubation to proceed for 1 hr to completely digest the DNA template. Do not continue this incubation longer than 2 hr. III.D. Nuclease Digestion to Remove DNA and ssrna 13

16 Silencer sirna Cocktail Kit (RNase III) E. Purification of dsrna This purification removes proteins, free nucleotides, and nucleic acid degradation products from the dsrna. For the quickest dsrna purification, preheat the Elution Solution to ~95 C before starting the purification procedure. 1. Add 10X Binding Buffer and 100% ethanol to the dsrna Amount Component 50 µl dsrna (from step D.2 above) 50 µl 10X Binding Buffer 150 µl Nuclease Free Water 250 µl 100% Ethanol Gently mix by pipetting. 2. Apply dsrna mixture to the Transcription Reaction Filter Cartridge, and draw it through Pipet the entire 500 µl dsrna mixture onto the filter in the Transcription Reaction Filter Cartridge, and draw it through by centrifugation or with a vacuum manifold. Centrifuge users: a. For each dsrna sample, place a Transcription Reaction Filter Cartridge in a Collection Tube. Use the Collection Tubes supplied with the kit b. Pipet the entire 500 µl dsrna mixture onto the filter in the Transcription Reaction Filter Cartridge. Centrifuge at maximum speed for 2 min. c. Discard the flow through and replace the Transcription Reaction Filter Cartridge in the Collection Tube. Vacuum manifold users: a. For each dsrna sample, place a 5 ml syringe barrel on the vacuum manifold, load it with a Transcription Reaction Filter Cartridge, and turn on the vacuum. b. Pipet the entire 500 µl dsrna mixture onto the filter in the Transcription Reaction Filter Cartridge. The vacuum will draw the lysate through the filter. 14 III.E. Purification of dsrna

17 Silencer sirna Cocktail Kit Protocol 3. Wash the Transcription Reaction Filter Cartridge with 2 X 500 µl Wash Solution 4. Recover the dsrna with two serial elutions in 50 µl hot Elution Solution The Elution Solution provided with the kit is 10 mm Tris-HCl ph 7, 1 mm EDTA. If desired, the dsrna can be eluted into any sterile low salt solution ( 30 mm). 5. Quantitation and storage of the dsrna 6. (optional) Check 1/400 th of the purified dsrna on an agarose gel Verify that 12 ml 100% ethanol was added to the 2X Wash Solution to make Wash Solution (section III.A on page 11). a. Pipet 500 µl of Wash Solution onto the filter in the Transcription Reaction Filter Cartridge. Draw the wash solution through the filter as in the previous step. b. Repeat with a second 500 µl aliquot of Wash Solution. c. After discarding the Wash Solution, continue centrifugation, or leave on the vacuum manifold for ~10 30 sec to remove the last traces of liquid. a. Transfer Transcription Reaction Filter Cartridge to a fresh Collection Tube. b. Apply 50 µl (hot) Elution Solution to the filter in the Transcription Reaction Filter Cartridge. Apply preheated ( 95 C) Elution Solution to the filter, or Apply room temperature Elution Solution, close the tube lid over the Transcription Reaction Filter Cartridge, and incubate in a heat block set to 65 C or warmer for 2 min. c. Centrifuge for 2 min at maximum speed. d. Repeat steps b c with a second 50 µl aliquot of Elution Solution collecting the RNA into the same Collection Tube. Most of the RNA will be eluted in the first elution. The second elution is included to recover any RNA that remains after the first elution. Quantitate the reaction product by measuring its absorbance at 260 nm and calculating the concentration (see section V.A. Quantitation of RNA by Spectrophotometry on page 26). The dsrna is stable when stored at 20 C in Elution Solution. Run 1/400 th of the dsrna on a 2% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation. 1/400 th of 100 µl elution volume is 2.5 µl of a 1:10 dilution Dilute the gel samples in TE (10 mm Tris, 1 mm EDTA) or in gel loading buffer (Instructions for running the gel are in section V.B.2 on page 27.) The dsrna will migrate slightly slower than DNA markers of the same length. III.E. Purification of dsrna 15

18 Silencer sirna Cocktail Kit (RNase III) F. RNase III Digestion and sirna Purification The Silencer sirna Cocktail Kit contains enough RNase III to digest a total of 600 µg of long dsrna into sirna. We typically digest 15 µg of long dsrna per RNase III reaction; this yields enough sirna for about 120 transfections in a 24 well dish at 50 nm final concentration. 1. Assemble RNase III digestion reaction in a microfuge tube Assemble the following reagents, and mix thoroughly. Amount Component up to 15 µg dsrna (up to 30 µl)* 15 µl RNase III 5µl 10X RNase III Buffer to 50 µl Nuclease-free Water * If your long dsrna concentration is <0.5 µg/µl, you have two choices: 1) Digest <15 ug by adding 30 µl of your long dsrna to the 50 µl reaction. 2) Use 15 µl RNase III and a final 1X RNase III Buffer concentration in a reaction that is large enough to accomodate 15 ug of your long dsrna. 2. Incubate 1 hr at 37 C Incubate the RNase III digestion reaction in a 37 C water bath for 1 hr. 3. Prewet the sirna Purification Unit, then purify the sirna by spinning it through a. Prewet the sirna Purification Unit by applying 50 µl of Nuclease-free Water to the top of the unit, and centrifuging at 14,000 X g for 8 min. b. Discard the collection tube from the bottom of the sirna Purification Unit, and insert the upper portion of the unit into a fresh Collection Tube (supplied with the kit). c. Load the RNase III reaction onto the center of an sirna Purification Unit and centrifuge at 14,000 X g for 8 min. As much as 500 µl or 150 µg of RNase III digested RNA can be purified on a single sirna Purification Unit. d. The undigested and partially RNase III digested material will be retained in the top of the sirna Purification Unit and the sirna will flow through the column into the tube. The sirna can be used directly for transfection. 4. Determine the concentration of the sirna Quantitate the reaction product by measuring its absorbance at 260 nm and calculating the concentration (see section V.A. Quantitation of RNA by Spectrophotometry on page 26). 16 III.F. RNase III Digestion and sirna Purification

19 Silencer sirna Cocktail Kit Protocol 5. (optional) Check the sirna on a 15% acrylamide gel If desired, analyze the quality of the sirna by running 1 2 µg of it on a 15% nondenaturing acrylamide gel. (See section V.B.3 on page 28 for instructions). See Figure 6 on page 20 for an example of how the sirna will look on a gel. G. sirna Cocktail Quantification The sirna cocktail concentration used for transfection is critical to the success of gene silencing experiments. Transfecting too much sirna causes nonspecific reductions in gene expression and toxicity to the transfected cells. Transfecting too little sirna does not change the expression of the target gene. Assuming that the UV spectrophotometer is accurate, measuring the absorbance of the sirna sample at 260 nm is the simplest method to assess the concentration of the sirna preparation. 1. Measure the A 260 of a 1:25 dilution of the sirna 2. Determine the concentration of the sirna cocktail in µg/ml 3. Determine the molar concentration of the sirna cocktail Dilute a small sample of the sirna 1:25 into TE (10 mm Tris-HCl ph 8, 1 mm EDTA) and read the absorbance at 260 nm in a spectrophotometer. Be sure to blank the spectrophotometer with the same TE that was used for sample dilution. Multiply the absorbance reading by 1,000 to determine the concentration of the purified sirna in µg/ml (explanation below). 1,000 = 25-fold dilution X 40 µg sirna/ml per absorbance unit Most Silencer sirna Cocktail Kit products are bp, so for the purpose of these calculations, we will use 13.5 bp as the size of the sirna. The molar concentration of the sirna in µm can be determined by dividing the µg/ml concentration of the sirna by 9 (explanation below). There are 9 µg of RNA in 1 nmole of a 13.5 bp dsrna: 13.5 nt X 2 strands = 27 nt X µg/nmol for each nt = 9 µg/nmol Dividing the µg/ml concentration by 9 yields the µm concentration as shown below: X µg ml 9 µg nmol = X µg ml X nmol 9 µg X nmol = = X µmol = X µm ml (9) L (9) 9 Therefore µm = X 9 III.G. sirna Cocktail Quantification 17

20 Silencer sirna Cocktail Kit (RNase III) 4. Example calculation A 1:25 dilution of purified sirna has an A 260 = 0.4. The molar concentration is determined as follows: 0.4 X 1,000 µg sirna/ml per A 260 = 400 µg/ml 400 µg/ml divided by 9 µg sirna/nmol sirna = ~44 µm sirna H. Transfecting Mammalian Cells The efficiency with which mammalian cells are transfected with sirna cocktails will vary according to cell type and the transfection agent used. This means that the optimal concentration used for transfections should be determined empirically. We have found that sirnas generated with the Silencer sirna Cocktail Kit typically work best when present in cell culture medium at nm, however a more extensive concentration range from nm can be analyzed in optimization experiments. Most protocols recommend maintaining mammalian cells in the medium used for transfection. This is to avoid diluting or removing the sirnas from the cells by adding medium or washing the cells with new medium. We have found that mammalian cells diluted 2 fold with fresh medium 24 hours after transfection typically exhibit greater viability than those left in the medium used for transfection. Furthermore, adding fresh medium does not appear to have a detrimental effect on the activity of the transfected sirnas. 18 III.H. Transfecting Mammalian Cells

21 Troubleshooting IV. Troubleshooting A. Use of the GAPDH Control Template The GAPDH Control Template is a linear GAPDH dsdna fragment with opposing T7 promoters. It will yield a 440 bp dsrna product. Once cleaved with RNase III, the dsrna is efficiently digested into an sirna cocktail that can be transfected into cells to knock down the expression of GAPDH. 1. Positive control reaction instructions a. Use 2 µl (200 ng) of GAPDH Control Template in a standard Silencer sirna Cocktail Kit reaction as described in section III.B starting on page 11. b. Incubate the transcription reaction (step III.B.4 on page 12) for 2 hr. c. Skip section III.C. Annealing RNA to Maximize Duplex Yield on page 12 because the sense and antisense RNA strands will hybridize during the transcription reaction. d. Follow the procedure as described in sections III.D and III.E starting on page 13 to purify the dsrna. In step III.E.4 on page 15, elute the dsrna with 2 X 50 µl Elution Solution. e. Measure the A 260 of the GAPDH dsrna reaction product as described in section V.A on page 26 to determine its concentration. The yield should be µg or more dsrna at this point (before digestion with RNase III). f. Digest 15 µg of the GAPDH dsrna and purify the sirna made following the instructions in section III.F on page 16. g. Measure the A 260 of the GAPDH sirna reaction product as described in section V.A on page 26 to determine its concentration. 4.5 µg of sirna for GADPH should be recovered after RNase III digestion and purification of 15 µg of GAPDH dsrna; this corresponds to at least 30% of the input dsrna. h. (optional) Run 1 2 µg of the positive control reaction product on a 15% acrylamide gel. The sirna should be bp in length, with the majority of the reaction products migrating at bp (see Figure 6 on page 20). IV.A. Use of the GAPDH Control Template 19

22 Silencer sirna Cocktail Kit (RNase III) 2. What to do if the positive control reaction doesn t work as expected If the yield of RNA from the control reaction is low, something is probably wrong either with the procedure or the kit, or the quantitation is in error. a. Double check the RNA quantitation When assessing yield by UV spectrophotometry, be sure to use TE (10 mm Tris-HCl, 1 mm EDTA) to blank the spectrophotometer and dilute the RNA. To confirm that the quantitation is correct, verify the yield by an independent method. For example if UV spectrophotometry was used to assess yield, try also running an aliquot of the reaction on an acrylamide gel and comparing its intensity to a sample of known concentration. b. Try the positive control reaction again If the yield is indeed low by two different measurements, there may be a technical problem with the way the kit is being used. For example, in the transcription reaction, the spermidine in the 10X T7 Reaction Buffer may cause precipitation of the template DNA if it is not diluted by the other ingredients prior to adding the DNA. (This is the reason that the water is added first.) Repeat the reaction, following the protocol carefully. If things still don t go well, contact Ambion s Technical Services ( , option 2) for more ideas. GAPDH control transcript chem. synthesized sirna Not RNase III treated RNase III treated + purif. 21 bp Figure 6. Positive Control Reaction Long dsrna made with the GAPDH Control Template was run on a 15% acrylamide gel before and after digestion with RNase III. Before RNase III treatment, the 440 bp transcript cannot even enter the gel; after digestion with RNase III, the majority of the sirna is bp long. The molecular weight marker is a chemically synthesized 21-mer dsrna. 20 IV.A. Use of the GAPDH Control Template

23 Troubleshooting B. Troubleshooting Low Yield from the Transcription Reaction The amount of RNA synthesized in a standard 20 µl transcription reaction should be µg or more; however, there is a great deal of variation in yield from different templates. If the yield is low, the first step in troubleshooting the reaction is to use the GAPDH Control Template in a standard Silencer sirna Cocktail Kit transcription reaction (section IV.A on page 19). 1. Neither my template nor the control reaction works 2. The control reaction works, but my template gives low yield a b Figure 7. Possible outcomes of mixing experiment 1 GAPDH Control Template 2 experimental template 3 mixture of 1 and 2 Double check that you have followed the procedure accurately, and consider trying the positive control reaction a second time. If the positive control still doesn t work, it is an indication that something may be wrong with the kit, call Ambion s Technical Support group for more ideas. If the transcription reaction with your template generates full-length, intact RNA, but the reaction yield is significantly lower than the amount of RNA obtained with the GAPDH Control Template, it is possible that contaminants in the DNA are inhibiting the RNA polymerase. A mixing experiment can help to differentiate between problems caused by inhibitors of transcription and problems caused by the sequence of a template. Include three reactions in the mixing experiment, using the following DNA templates: 1. 2 µl GAPDH Control Template µg experimental DNA template 3. a mixture of 1 and 2 Assess the results of the mixing experiment by running 0.5 µl of the transcription reaction on an agarose gel as described in section V.B.2 on page 27. a. Transcription of the GAPDH Control Template is inhibited by your template. (See Figure 7.a) This implies that inhibitors are present in your DNA template. Typical inhibitors include residual SDS, salts, EDTA, and RNases. Proteinase K treatment frequently improves template quality. Treat template DNA with Proteinase K ( µg/ml) and SDS (0.5%) for 30 min at 50 C, followed by phenol/chloroform extraction and ethanol precipitation. Carry-over of SDS can be minimized by diluting the nucleic acid several fold before ethanol precipitation, and excess salts and EDTA can be removed by vigorously rinsing nucleic acid pellets with 70% ethanol before resuspension. IV.B. Troubleshooting Low Yield from the Transcription Reaction 21

24 Silencer sirna Cocktail Kit (RNase III) b. Adding your template to the reaction with the GAPDH Control Template does not inhibit synthesis of the control RNA. (See Figure 7.b.) This indicates that the problem may be inherent to your template. i. Check the amount and quality of template Another possibility is that the template quantitation is inaccurate. If quantitation was based on UV absorbance and the DNA prep had substantial amounts of RNA or chromosomal DNA, the amount of template DNA may be substantially less than the calculated value. Also, check an aliquot of the template DNA on an agarose gel to make sure it is intact and that it is the expected size. If there is even a small amount of circular template in the transcription reaction it will reduce the yield of dsrna (this is discussed in section II.D.2 on page 9). ii. Use the maximum recommended template amount If you used less than the maximum amount of template in the transcription reaction; 500 ng of <500 bp templates, or 1 µg of >500 bp templates, then increasing the template amount to these levels may increase RNA yield. If you are transcribing sense and antisense RNA from two separate templates, use 500 ng to 1 µg of each template in the reaction. iii. Extend the transcription reaction time Another parameter that can be adjusted is reaction time. Extending the standard 2 hr incubation to 6 10 hr or even overnight may improve yield. iv. Change your priming region Some sequences are simply inefficient transcription templates. If low RNA yield results even after checking the template, and trying overnight incubation of the transcription reaction, it may be necessary to prepare a transcription template from a different region of the gene. Often simply moving the transcription start point can overcome problems with inefficient transcription; typically there are several regions of the gene that will transcribe with equal efficiency. Also, transcription efficiency may be higher when the transcription template contains 2 3 bases of purines immediately following the GGG sequence at positions +1 to +3 in the T7 promoter sequence (T7 promoter sequence is shown in Figure 1 on page 7). 22 IV.B. Troubleshooting Low Yield from the Transcription Reaction

25 Troubleshooting v. Use 2 separate templates Sometimes the template will simply not transcribe well with opposing promoters, and the two strands of RNA need to be made from separate templates and annealed after synthesis. (If the template was made by PCR, this will only require 2 additional PCR primers.) C. Troubleshooting Unexpected Transcription Reaction Products 1. Transcription reaction products produce a smear when run on a gel 2. Transcription reaction products run as more than one band, or as a single band smaller than expected This problem is usually only seen with single-strand transcriptions. If the RNA appears degraded (e.g. smeared), remove residual RNase from the DNA template preparation before in vitro transcription. Do this by digesting the DNA prep with proteinase K ( µg/ml) in the presence of 0.5% SDS for 30 min at 50 C, follow this with phenol/chloroform extraction. The RNase Inhibitor (SUPERase In) in the transcription reaction, can only inactivate moderate RNase contamination. Large amounts of RNase in the DNA template will compromise the size and amount of transcription products. Premature termination of transcription If gel analysis shows multiple discrete bands or a single band smaller than the expected size, there may be problems with premature termination by the polymerase. Even if transcription of only one of the strands was prematurely terminated, the single-stranded portion of the duplex will be digested during the nuclease treatment resulting in a shorter than expected dsrna. Possible causes of premature termination are sequences which resemble the phage polymerase termination signals, stretches of a single nucleotides, and GC-rich templates. Termination at single polynucleotide stretches can sometimes be alleviated by decreasing the transcription reaction temperature (Krieg PA 1990). We suggest testing 30 C, 20 C and 10 C, but be sure to increase the reaction time to offset the decrease in yield caused by incubation at temperatures <37 C. There is a report that single-stranded binding (SSB) protein increased the transcription efficiency of a GC rich template (Aziz and Soreq, 1990). IV.C. Troubleshooting Unexpected Transcription Reaction Products 23

26 Silencer sirna Cocktail Kit (RNase III) 3. Transcription reaction products are larger than expected Silencer sirna Cocktail Kit products occasionally run as two bands after the nuclease digestion and dsrna purification; one at the expected size, and one that is double the expected size. If this occurs, check the size of the transcription template on a gel to verify that it is pure and sized correctly. Multi-strand aggregates are present in the mixture Larger than expected bands or ethidium bromide staining in the wells could be seen as a result of aggregates of multiple RNA strands. These can be denatured by heating the solution to C for ~3 min, and then leaving it at room temperature until it reaches ambient temperature. Be sure that RNA solutions are in a solution containing at least 1 mm EDTA (such as the Elution Solution supplied with the kit) for the heat treatment. D. Troubleshooting RNase III Digestion and sirna Purification 1. My dsrna is not digested by RNase III a. Check that the RNase III is active by doing the positive control reaction (section IV.A on page 19) Check to see if the dsrna transcribed from the GAPDH Control Template is digested by running 1 2 µg of the sirna on a 15% acrylamide gel as described in section V.B.3 on page 28. The reaction products should be bp. If the positive control reaction does not yield the expected products, troubleshoot as described in section A.2 on page 20. b. RNase III will not fully digest single-stranded RNA (ssrna) RNase III cuts dsrna, so if your long dsrna contains a significant amount of ssrna, it will only be clipped in regions where secondary structure makes it double-stranded. Troubleshoot your transcription to obtain good yield of both strands of dsrna (suggestions in sections IV.B starting on page 21, and IV.C starting on page 23). If the dsrna was produced from two separate transcription templates (in a single transcription reaction, or in two separate reactions), be sure to anneal the two ssrna to produce dsrna as described in section III.C starting on page 12. It is possible that the RNase A in the nuclease digestion (section III.D on page 13) did not remove all of the ssrna. You can continue the digestion for up to 2 hr to maximize ssrna removal. 24 IV.D. Troubleshooting RNase III Digestion and sirna Purification