silencer™ sirna cocktail kit (rnase iii) · silencer™ transfection kit cat #1630 the silencer...

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

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1A. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6A. Choosing the Target Site

B. Strategies for Transcription of dsRNAC. PCR Templates

D. Plasmid Templates

III. Silencer siRNA Cocktail Kit Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11A. 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 ssRNAE. Purification of dsRNA

F. RNase III Digestion and siRNA Purification

G. siRNA Cocktail Quantification

H.Transfecting Mammalian Cells

IV. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19A. Use of the GAPDH Control Template

B. Troubleshooting Low Yield from the Transcription ReactionC. Troubleshooting Unexpected Transcription Reaction Products

D. Troubleshooting RNase III Digestion and siRNA Purification

V. Additional Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26A. Quantitation of RNA by Spectrophotometry

B. Agarose and Acrylamide Gel Electrophoresis Instructions

VI. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30A. References

B. Silencer siRNA Cocktail Kit Specifications

C. Aqueous Solution Material Safety Data Sheet

Manual Version 0305Literature 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 Technol-ogy to US Patent Applications 60/265232, 09/821832 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, 650-723-0651. This is STANFORD reference S02-028.

Warranty and Liability Ambion is committed to providing the highest quality reagents at compet-itive 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.

Introduction

I. Introduction

A. BackgroundThe Silencer™ siRNA Cocktail Kit (RNase III) (patents pending) is a system for in vitro transcription of long dsRNA and its enzy-matic 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 tech-nology 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)

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), trypa-nosomes (Ngo et al. 1998), planaria (Sánchez-Alvarado and New-mark 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 dou-ble-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 21–23 bp in length and contain 3' dinucle-otide 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 (Ham-mond 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.

Cell Membrane

RISC??

RNase

21–23 bpsiRNAs

siRNAs associatewith RISC complex

Cleavage of mRNAby RISC

dsRNA cleavage

Degradation by exonucleases

?

long dsRNA

RNA Interference in vivo

I.A. Background 1

Silencer™ siRNA Cocktail Kit (RNase III)

Silencer siRNA Cocktail Kit procedure overview

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 reac-tion to produce siRNA. The recombinant E. coli RNase III used for this reaction cleaves long dsRNA into short 12–30 bp dsR-NAs containing a 5'-PO4, 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 Purifica-tion Units to remove any undigested or partially digested mate-rial. After purification, the siRNA can be used for inducing RNAi by introduction into mammalian cells. We and others have dem-onstrated that siRNAs generated using bacterial RNase III are capable of efficiently and specifically inducing RNAi in mamma-lian cells (Yang et al. 2002, Calegari et al. 2002, Byrom et al. 2003, Trotta et al. in press).

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 Cock-tail Kit—or Ambion’s Silencer siRNA Construction Kit), and using siRNA expression vectors (e.g. Ambion’s pSilencer™ vec-tors). 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.

Nuclease Digestion (37˚C)1 hr

RNase III Digestion (37˚C)1 hr

dsRNA Purification10–15 min

siRNA Purification10 min

Transfection

Transcription2– 4 hr

5' 3'

5' 3'

5' 3'

2 I.A. Background

Introduction

B. Reagents Provided with the Kit and StorageThe 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 temp60 Collection Tubes room temp20 siRNA Purification Units room temp40 µl T7 Enzyme Mix

T7 RNA polymerase, SUPERase•In, and other components in buffered 50% glycerol

–20°C

40 µl 10X T7 Reaction Buffer –20°C40 µl ATP Solution (75 mM) –20°C40 µl CTP Solution (75 mM) –20°C40 µl GTP Solution (75 mM) –20°C40 µl UTP Solution (75 mM) –20°C1 ml 10X Binding Buffer –20°C

10 ml Nuclease-free Water –20°C4 ml Elution Solution –20°C

40 µl RNase A –20°C600 µl RNase III (1U/µl) –20°C500 µl 10X RNase III Buffer –20°C45 µl DNase I –20°C

100 µl 10X Digestion Buffer –20°C12 ml 2X Wash Solution

Add 12 ml 100% ethanol before use–20°C

10 µl GAPDH Control Template (100 ng/µl) –20°C

I.B. Reagents Provided with the Kit and Storage 3


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 corre-sponding 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 Reac-tion 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 Reac-tion Filter Cartridges.

To assess the reaction products:

Reagents and equipment for acrylamide gel electrophoresis

Spectrophotometer


Silencer™ Transfection KitCat #1630

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.

RNase IIICat #2290

Escherichia coli Ribonuclease III (RNase III; EC 3.1.24) is a dou-ble-stranded RNA (dsRNA) specific endoribonuclease. It cleaves dsRNA into 12–15 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 mamma-lian cells (patent pending).

siRNA Purification UnitsCat #10074G

These are the same siRNA Purification Units as those used in the SilencersiRNA 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.

Silencer™ siRNA Labeling KitCat #1632, 1634

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

Introduction

Silencer™ siRNA ControlsCat #4602–4606

The Silencer siRNA Controls are ready-to-use, chemically synthesized, purified GAPDH, Cyclophilin, or c-myc siRNA for up to 150 transfec-tions (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.

Negative Control siRNAsee Catalog or Web site

Universal scrambled siRNA control sequences are available separately as either prepared and tested siRNA or templates for use in the SilencersiRNA 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.

RNaseZap®Cat #9780–84

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.

Electrophoresis Reagentssee Catalog or Web site

Ambion offers gel loading solutions, agaroses, acrylamide solutions, pow-dered gel buffer mixes, nuclease-free water, and RNA and DNA molecu-lar weight markers for electrophoresis. Please see our catalog or our web site (www.ambion.com) for a complete listing as this product line is always growing.

I.D. Related Products Available from Ambion 5

http://www.ambion.com


II. Preparation of Template DNA

A. Choosing the Target SiteExperiments at Ambion using the Silencer siRNA Cocktail Kit have not shown a difference in the effectiveness of siRNA cocktails tar-geting 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 450–1365 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 150–500 bp in length provide the highest level of RNAi. With these smaller dsRNA substrates, the RNase III digestion yields an siRNA cock-tail with fewer unique siRNAs, but each one is at a higher con-centration 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, 150–500 bp dsRNAs are extremely likely to yield an siRNA cocktail that effectively induces RNAi.

B. Strategies for Transcription of dsRNARNase III requires a dsRNA template. Since the T7 RNA poly-merase 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 reac-tion 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 tran-scribed. 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 dic-tated 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

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.

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 SilencersiRNA Cocktail Kit transcription reaction. Synthesize PCR prim-ers 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.

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.

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.

5'-TAATACGACTCACTATAGGGAGA-3'+1

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 promoterappended to both PCR primers

• 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 anneal-ing step will not be needed after the tran-scription reaction; dsRNA will form during the transcription reaction.

T7 gene specific

T7gene specific gene specific

gene specific T7 gene specific

T7gene specific

II.C. PCR Templates 7


• 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 calcu-lated Tm 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 anneal-ing events can use the entire primer site; therefore use the cal-culated Tm 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.

3. Check PCR products on a gel before using them in this procedure

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.

4. Purification of the PCR products

If the PCR yields multiple bands on a gel, it may be necessary to gel purify the correct size PCR product using standard purifica-tion techniques. Note that optimizing the PCR conditions may result in amplification of a single band.

Figure 3. Strategy for Adding a Single T7 Promoter by PCR

RNA Transcript

PCR Primer

+1

5'-TAATACGACTCACTATAGGGTACTTGCATTACCCCTCGA-3'

5'-TGCATTACCCCTCGAATT...GTGATCAGATGCTAGGTAC-3'3'-ACGTAATGGGGAGCTTAA...CACTAGTCTACGATCCATG-5'

5'-TAATACGACTCACTATAGGGTACTTGCATTACCCCTCGAATT...GTGATCAGATGCTAGGTAC-3' 3'-ATTATGCTGAGTGATATCCCATGAACGTAATGGGGAGCTTAA...CACTAGTCTACGATCCATG-5'

5'-pppGGGUACUUGCAUUACCCCUCGAAUU...GUGAUCAGAUGCUAGGUAC-3'

3'-AGTCTACGATCCATG-5'

Target DNA

T7 PCR primer

T7 promoter

+1

PCR

Trxn

8 II.C. PCR Templates

Preparation of Template DNA

D. Plasmid Templates

1. Cloning strategy In general, to use plasmid templates in the Silencer siRNA Cock-tail Kit, it is best to make two separate clones with the same target region in both orientations.

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 frag-ment during PCR, then ligate the PCR product into a cloning vector via the restriction sites.

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 tem-plate, 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 cir-cular plasmid in a template prep will generate a large proportion of transcript, wasting much of the synthetic capacity of the reaction.

Figure 4. Cloning in plasmids

T 7 Promoter T 7 Promoter

We routinely use all types of restriction enzymes, however, there has been one report of low level transcription from the inap-propriate template strand in plas-mids cut with restriction enzymes leaving 3' overhanging ends (pro-duced by Kpn I, Pst I, etc.; Schen-dorn and Mierindorf, 1985).

Figure 5. Linearized plasmids

T 7

T 7

II.D. Plasmid Templates 9


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 NH4OAc

• 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 0.5–1 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 prep-aration systems yield DNA that works well in the Silencer siRNA Cocktail Kit.

Note that DNA from some miniprep procedures may be contami-nated with residual RNase A. Also, restriction enzymes occasion-ally 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 (100–200 µg/ml) and 0.5% SDS for 30 min at 50°C, follow this with phenol/chloro-form extraction (using an equal volume) and ethanol precipitation.

10 II.D. Plasmid Templates

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

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.

2. Assemble transcription reaction at room temperature

• 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


The following amounts are for a single 20 µl transcription reac-tion. Reactions may be scaled up or down if desired.

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 YieldAnnealing the complementary RNA is often unnecessary for tran-scripts ≤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 aggre-gates (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 synthe-sis reactions.

• Include this annealing step for production of ≤800 nt dsRNA when the two strands were synthesized from sepa-rate transcription templates (in the same or in separate transcription reactions).

Amount Component EGAscript

to 20 µl Nuclease-free Water0.2– 2 µg Linear template DNA*

2 µl 10X T7 Reaction Buffer2 µl ATP Solution2 µl CTP Solution2 µl GTP Solution2 µl UTP Solution2 µl T7 Enzyme Mix

* Templates


1. Mix the transcription reactions containing complementary RNA

If sense and antisense RNA was synthesized in a single transcrip-tion 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.

2. Incubate at 75° for 5 min, then cool to room temperature

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.

3. (optional) Check 1/400th of the dsRNA on an agarose gel

Run 1/400th of the dsRNA on a 1% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation.

• 1/400th of a 20 µl dsRNA solution is 5 µl of a 1:100 dilution

• 1/400th 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 ssRNAThis 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

The amounts shown are for a 20 µl transcription reaction; scale up if your transcription reaction was larger.

2. Incubate at 37°C for 1 hr

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.

Amount Component

20 µl dsRNA (from step B.4 or step C.2)21 µl Nuclease-free Water5 µl 10X Digestion Buffer2 µl DNase I2 µl RNase A

III.D. Nuclease Digestion to Remove DNA and ssRNA 13


E. Purification of dsRNAThis 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

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.

Amount Component

50 µl dsRNA (from step D.2 above)50 µl 10X Binding Buffer

150 µl Nuclease Free Water250 µl 100% Ethanol

14 III.E. Purification of dsRNA


3. Wash the Transcription Reaction Filter Cartridge with 2 X 500 µl Wash Solution

Verify that 12 ml 100% ethanol was added to the 2X Wash Solu-tion 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.

4. Recover the dsRNA with two serial elutions in 50 µl hot Elution Solution

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

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).

• 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.

5. Quantitation and storage of the dsRNA

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.

6. (optional) Check 1/400th of the purified dsRNA on an agarose gel

Run 1/400th of the dsRNA on a 2% agarose gel (nondenaturing) to examine the integrity and efficiency of duplex formation.

• 1/400th 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


F. RNase III Digestion and siRNA PurificationThe 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.

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. Quan-titation of RNA by Spectrophotometry on page 26).

Amount Component

up to 15 µg dsRNA (up to 30 µl)*

* If your long dsRNA concentration is


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 QuantificationThe 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 A260 of a 1:25 dilution of the siRNA

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 spectropho-tometer with the same TE that was used for sample dilution.

2. Determine the concentration of the siRNA cocktail in µg/ml

Multiply the absorbance reading by 1,000 to determine the con-centration of the purified siRNA in µg/ml (explanation below).

3. Determine the molar concentration of the siRNA cocktail

Most Silencer siRNA Cocktail Kit products are 12–15 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:

• Dividing the µg/ml concentration by 9 yields the µM concen-tration as shown below:

1,000 = 25-fold dilution X 40 µg siRNA/ml per absorbance unit

13.5 nt X 2 strands = 27 nt X 0.333 µg/nmol for each nt = 9 µg/nmol

X µg

ml

nmol

9 µg=

X µgml

X nmol9 µg

=ml (9)X nmol = X µmol

L (9)=

X µM9

Therefore µM = X ÷ 9

III.G. siRNA Cocktail Quantification 17


4. Example calculation A 1:25 dilution of purified siRNA has an A260 = 0.4. The molar concentration is determined as follows:

H. Transfecting Mammalian CellsThe efficiency with which mammalian cells are transfected with siRNA cocktails will vary according to cell type and the transfec-tion 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 50–100 nM, however a more extensive concentration range from 1–100 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 remov-ing 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 typi-cally 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 trans-fected siRNAs.

0.4 X 1,000 µg siRNA/ml per A260 = 400 µg/ml

400 µg/ml divided by 9 µg siRNA/nmol siRNA = ~44 µM siRNA

18 III.H. Transfecting Mammalian Cells

Troubleshooting

IV. Troubleshooting

A. Use of the GAPDH Control TemplateThe GAPDH Control Template is a linear GAPDH dsDNA frag-ment 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 Yieldon 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.Estarting 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 A260 of the GAPDH dsRNA reaction product as described in section V.A on page 26 to determine its concentration. The yield should be 30–40 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 A260 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 12–30 bp in length, with the majority of the reaction products migrating at 12–15 bp (see Figure 6 on page 20).

IV.A. Use of the GAPDH Control Template 19


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 quan-titation 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 spectro-photometer and dilute the RNA.

• To confirm that the quantitation is correct, verify the yield by an independent method. For example if UV spectrophotome-try 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 againIf 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 (800-888-8804, option 2) for more ideas.

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 12–15 bp long. The molecular weight marker is a chemically synthesized 21-mer dsRNA.

chem

. syn

thes

ized

siR

NA

GAPDH controltranscript

RN

ase

III t

reat

ed +

pur

if.

21 bp –

Not

RN

ase

III t

reat

ed

20 IV.A. Use of the GAPDH Control Template

Troubleshooting

B. Troubleshooting Low Yield from the Transcription ReactionThe amount of RNA synthesized in a standard 20 µl transcription reaction should be 30–40 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

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.

2. The control reaction works, but my template gives low yield

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 Con-trol 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 transcrip-tion 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 Template2. 1–2 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 sec-tion 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 (100–200 µg/ml) and SDS (0.5%) for 30 min at 50°C, fol-lowed by phenol/chloroform extraction and ethanol precipita-tion. 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.

1 2 3

1 2 3

Figure 7. Possible outcomes of mixing experiment

a

b

1 – GAPDH Control Template 2 – experimental template 3 – mixture of 1 and 2

IV.B. Troubleshooting Low Yield from the Transcription Reaction 21


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 templateAnother 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 amountIf you used less than the maximum amount of template in the transcription reaction; 500 ng 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 timeAnother 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 regionSome sequences are simply inefficient transcription tem-plates. If low RNA yield results even after checking the template, and trying overnight incubation of the transcrip-tion 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 prob-lems with inefficient transcription; typically there are sev-eral 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 posi-tions +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

Troubleshooting

v. Use 2 separate templatesSometimes 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

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 tran-scription. Do this by digesting the DNA prep with proteinase K (100–200 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.

2. Transcription reaction products run as more than one band, or as a single band smaller than expected

Premature termination of transcriptionIf 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 prema-turely 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 some-times 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 temper-atures


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 mixtureLarger 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 75–100°C for ~3 min, and then leaving it at room tempera-ture 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 Con-trol 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 12–30 bp. If the positive control reaction does not yield the expected products, trouble-shoot 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 signif-icant 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 start-ing 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 sepa-rate 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

Troubleshooting

• It may also be a good idea to look at the long dsRNA on an agarose gel to make sure that it is the expected size.

2. My RNA concentration is drastically reduced after purification on an siRNA Purification Unit

The siRNA Purification Unit will retain undigested dsRNA, therefore problems with these units are very likely to actually be problems with the RNase III digestions (see troubleshooting sug-gestions above).

IV.D. Troubleshooting RNase III Digestion and siRNA Purification 25


V. Additional Procedures

A. Quantitation of RNA by SpectrophotometryThe concentration of dsRNA can be determined by diluting an aliquot of the preparation (usually a 1:10 to 1:25 dilution) in TE (10 mM Tris-HCl pH 8, 1 mM EDTA), and reading the absor-bance in a spectrophotometer at 260 nm. The buffer used for dilution need not be RNase-free (unless you want to recover the RNA), since slight degradation of the RNA will not significantly affect its absorbance. The concentration of RNA in µg/ml can be calculated as follows:

1 A260 = 40 µg RNA/mlso, A260 x dilution factor x 40 = µg/ml RNA

B. Agarose and Acrylamide Gel Electrophoresis Instructions

1. Solutions for gel electrophoresis

a. 10X TBETBE is generally used at 1X final concentration for preparing gels and/or for gel running buffer.

Dissolve with stirring in about 850 ml nuclease-free water. Adjust the final volume to 1 L.Alternatively, Ambion offers 10X TBE as a ready-to-resuspend mixture of ultrapure molecular biology grade reagents (Ambion Cat #9863). Each packet makes 1 L of 10X TBE.

Concentration Component for 1 L0.9 M Tris base 109 g0.9 M Boric Acid 55 g20 mM 0.5 M EDTA solution 40 ml

26 V.A. Quantitation of RNA by Spectrophotometry

Additional Procedures

b. 6X non-denaturing gel loading buffer

Alternatively, Ambion offers an all-purpose Gel Loading Solu-tion for native agarose and acrylamide gels: Cat #8556. This 10X solution is rigorously tested for nuclease contamination and functionality.

2. Pouring and running agarose gels

a. Options for staining with ethidium bromideThere are several different ways to stain agarose gels, they are equivalent in terms of efficacy, but no two researchers can agree which is the most convenient.

• Add 0.5 µg/ml ethidium bromide to the gel mix, the run-ning buffer, or both.

• Add 10 µg/ml ethidium bromide to the gel loading buffer. All the samples should have the same amount of ethidium bromide, because it will affect the electrophoretic mobility of nucleic acids.

b. Prepare agarose gel mix, and pour the gel

i. Pour a volume of 1X TBE corresponding to the volume of gel needed into a flask with a capacity 5–10 fold the gel volume.

ii. Weigh 2 g agarose per 100 ml of gel needed, and add it to the 1X TBE. Heat with intermittent swirling until the agarose is completely melted.

iii. Leave at room temp to cool to ~65°C. While the gel is cooling, prepare the gel mold.

iv. When the agarose has cooled so that touching the flask is tolerable, slowly pour the agarose into the mold. Place the comb, and pop any bubbles that have formed with a clean pipet tip or a heated needle, and let the gel solidify.

Concentration Component for 10 ml

37 % glycerol (100%) 3.7 ml0.025 % bromophenol blue 2.5 mg0.025 % xylene cyanol 2.5 mg

20 mM 1 M Tris-HCl, pH 8 200 µl5 mM 500 mM EDTA 100 µl

nuclease-free water to 10 ml

V.B. Agarose and Acrylamide Gel Electrophoresis Instructions 27


c. Prepare samples and run the agarose gel

i. Put 0.5 µl of each sample into a fresh tube, and add gel loading buffer to samples for a final concentration of 1X. Flick or vortex the tubes to mix.

ii. Place the gel in the electrophoresis chamber and fill it with running buffer (1X TBE) to cover the gel completely. Remove the comb from the gel carefully.

iii. Check that the wells are intact and free of debris, and load the samples into separate wells. It is convenient to load a molecular weight marker to identify bands after the electrophoresis.

iv. Run the gel at 3.5–5.5 volts/cm until the bromophenol blue (the faster migrating dye) travels 1/2 to 2/3 the length of the gel.

v. View the gel on a UV transilluminator.

3. Pouring and running acrylamide gels

a. Prepare the acrylamide gel mix, and pour the gel

i. Assemble 15% nondenaturing acrylamide gel mix (the 15 ml recipe shown is enough for one 13 cm X 15 cm X 0.75 mm gel)

ii. Stir to mix, and then add:

iii. Mix briefly after adding the last two ingredients and pour gel immediately.

iv. Place the comb, and allow the gel to set.

b. Prepare siRNA samples, and run the gel

i. Put 1–2 µl of each siRNA sample into a fresh tube, and add gel loading buffer to a final concentration of 1X. Flick or vortex the tubes to mix.

ii. Place the gel in the electrophoresis chamber and fill the chambers with 1X TBE to cover the top of the gel. Remove the comb from the gel carefully.

Amount Component

1.5 ml 10X TBE4.75 ml 40% acrylamide (acrylamide:bis = 19:1)8.61 ml nuclease-free water

120 µl 10% ammonium persulfate16 µl TEMED

28 V.B. Agarose and Acrylamide Gel Electrophoresis Instructions

Additional Procedures

iii. Load the samples into separate wells of the gel. (Chemically synthesized 21-mer siRNA makes a good size marker.)

iv. Nondenaturing gels must be run slowly to avoid heat denaturation of the samples. The voltage should be set to ~8.3V/cm; a 13 X 15 cm X 0.75 mm gel should be run at about 100 V for ~2–3 hours.

v. Stain the gel in water or 1X TBE containing 400 ng/ml ethidium bromide for ~10 min at room temp with gentle agitation.

vi. View the gel on a UV transilluminator.

V.B. Agarose and Acrylamide Gel Electrophoresis Instructions 29


VI. Appendix

A. ReferencesAziz RB and Soreq H (1990) Improving poor in vitro transcription from GC-rich genes. Nucl. Acids Res. 18: 3418.

Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Role for a bidentate ribonu-clease in the initiation step of RNA interference. Nature, 409, 363-366.

Browning KS (1989) Transcription and translation of mRNA from polymerase chain reaction-gener-ated DNA. Amplifications 3: 14–15.

Byrom MW, Chang AM, Ford LP (2003) Inducing RNAi with siRNA cocktails generated by RNase III. Ambion TechNotes 10(1): 4–6

Calegari F, Haubensak W, Yang D, Huttner WB, Buchholz F. 2002. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci USA Oct 29;99(22):14236-40

Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001c) RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev,. 15, 188-200

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391(6669):806–11.

Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G and Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small tem-poral RNAs that control C. elegans developmental timing. Cell, 106, 23-34.

Hamilton AJ and Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 286, 950-952.

Knight SW and Bass BL (2001) A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science, 293, 2269-2271

Krieg PA and Melton DA (1987) In vitro RNA synthesis with SP6 RNA polymerase. Meth. Enzymol. 155: 397–415.

Krieg PA (1990) Improved Synthesis of Full-Length RNA Probes at Reduced Incubation Tempera-tures. Nucl. Acids Res. 18: 6463.

Lohmann JU, Endl I, Bosch TC. (1999) Silencing of developmental genes in Hydra. Dev Biol. Oct 1;214(1):211–4.

Milligan JF, Groebe DR, Witherell GW, and Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA template. Nucl. Acids Res. 15: 8783–8798.

30 VI.A. References

Appendix

Misquitta L, Paterson BM (1999) Targeted disruption of gene function in Drosophila by RNA inter-ference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA. 96(4):1451–6.

Molecular Cloning, A Laboratory Manual, 2nd edition. (1989) editor C Nolan, Cold Spring Harbor Laboratory Press.

Montgomery MK, Xu S, Fire A (1998) RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA. 95(26):15502–7.

Morris JC, Wang Z, Drew ME, Paul KS, Englund PT. (2001) Inhibition of bloodstream form Trypa-nosoma brucei gene expression by RNA interference using the pZJM dual T7 vector. Mol Biochem Parasitol. 117(1):111–3.

Mullis KB, and Faloona F (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Meth. Enzymol. 155: 335–350.

Nishikura K (2001) A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 16:415–418.

Ngo H, Tschudi C, Gull K, Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA. 95(25):14687–92.

Rubin GM, Spradling AC. (1982) Genetic transformation of Drosophila with transposable element vectors. Science. Oct 22;218(4570):348-53.

Sanchez Alvarado A, Newmark PA (1999) Double-stranded RNA specifically disrupts gene expres-sion during planarian regeneration. Proc Natl Acad Sci USA. 96(9):5049–54.

Schenborn ET and Mierindorf RC (1985) A novel transcription property of SP6 and T7 RNA poly-merases: dependence on template structure. Nucl. Acids Res. 13: 6223–6236.

Stoflet ES, Koeberl DD, Sarkar G, and Sommer SS (1988) Genomic amplification with transcript sequencing. Science 239: 491–494.

Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature. 395(6705):854.

Trotta R, Vignudelli T, Pecorari L, Guerzoni C, Santilli G, Candini O, Byrom M, Goldoni S, Intine RV, Maraia RJ, Ford LP, Caligiuri MA, Perrotti D, Calabretta B (In Press) BCR/ABL activates mdm2 mRNA translation via the La antigen in advanced Chronic Myelogenous Leukemia. Cancer Cell.

Wargelius A, Ellingsen S, Fjose A (1999) Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochem Biophys Res Commun. 263(1):156–61.

Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, Bishop JM (2002) Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci USA Jul 23;99(15):9942-7

Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1):25–33.

VI.A. References 31


32 VI.A. References

Appendix

B. Silencer siRNA Cocktail Kit Specifications

Kit contents and storage:

Stability: Store the kit components at the temperatures indicated above.

Properly stored kits are guaranteed for 6 months from the date received.

Quality Control Functional Testing

The GAPDH Control Template (200 ng) is used in a SilencersiRNA Cocktail Kit reaction as described in section IV.A on page 19, and is shown to produce ≥50 µg of ~440 bp dsRNA.

Nuclease testing

Each component is tested in Ambion’s rigorous nuclease assays.

Amount Component Storage

20 Transcription Rxn Filter Cartridges room temp60 Collection Tubes room temp20 siRNA Purification Units room temp40 µl T7 Enzyme Mix –20°C40 µl 10X T7 Reaction Buffer –20°C40 µl ATP Solution (75 mM) –20°C40 µl CTP Solution (75 mM) –20°C40 µl GTP Solution (75 mM) –20°C40 µl UTP Solution (75 mM) –20°C1 ml 10X Binding Buffer –20°C

10 ml Nuclease-free Water –20°C4 ml Elution Solution –20°C

40 µl RNase A –20°C600 µl RNase III (1U/µl) –20°C500 µl 10X RNase III Buffer –20°C45 µl DNase I –20°C

100 µl 10X Digestion Buffer –20°C12 ml 2X Wash Solution

Add 12 ml 100% ethanol before use–20°C

10 µl GAPDH Control Template (100 ng/µl) –20°C

VI.B. Silencer siRNA Cocktail Kit Specifications 33


RNase activityNone detected after incubation with 32P-labeled RNA; ana-lyzed by PAGE.

Non-specific endonuclease/nickase activityNone detected after incubation with supercoiled plasmid DNA; analyzed on 1% agarose.

Exonuclease activityNone detected after incubation with 32P-labeled Sau3A frag-ments of pUC19; analyzed by PAGE.

Protease testing

None detected in protein-containing components after incuba-tion with protease substrate, and analysis by spectrophotometry.

Safety Information:

This kit contains dilute aqueous solutions none of which are thought to present any health hazards. An MSDS for these com-ponents is supplied on the following pages.

34 VI.B. Silencer siRNA Cocktail Kit Specifications

Appendix


Physical data

Appearance and odor clear liquid, odorless

Boiling point n/a

Specific gravity 1.0 cm3 (about the same as water)

Solubility in H2O soluble

Fire and explosion hazard data

Flash point does not flash

Flammable limits in air no data

Extinguishing media water, CO2, foam, dry chemical

Special fire fighting Wear self-contained breathing apparatus and protective clothing

Fire/explosion hazards Irritating and/or toxic gases or fumes may be generated by thermal decomposition or combustion.

Health hazard data

Effects of overexposure Irritant, may be harmful by inhalation or ingestion.

Emergency first aid Wash affected area with water. Irrigate eyes for ≥15 minutes. See physician immediately. If ingested, drink several glasses of water, induce vomiting.

Reactivity data

Stability stable

Incompatibility n/a

Hazardous decomp. products n/a

Hazardous polymerization will not occur

Spill or leak procedures

If released or spilled Ventilate area. Absorb spill with inert material. Place in container with a lid. Wash spill area when cleanup is complete.

Waste disposal method Dispose according to federal, local and state regulations.

Special protection and precaution information

Respiratory protection Not expected to require personal respirator usage. (Use NIOSH approved respirator if necessary)

Ventilation Not expected to require any special ventilation.

Precautionary labeling none

Handling and storage considerations Laboratory aprons and gloves. Do not store in aluminum or copper con-tainers. Keep tightly closed in a cool, dry place.

This bulletin is for your guidance and is based upon information and tests believed to be reliable. Ambion makes no guarantee of the accuracy or completeness of the data and shall not be liable for any damages thereto. The data are offered solely for your consideration, investigation, and verification. These suggestions should not be confused with either state, municipal, or insur-ance requirements, or with national safety codes and constitute no warranty. Any use of these data and information must be determined by the user to be in accordance with applicable federal, state, and local regulations.

VI.C. Aqueous Solution Material Safety Data Sheet 35

I. IntroductionA. BackgroundRNA interference (RNAi) and short interfering RNAs (siRNAs)Silencer siRNA Cocktail Kit procedure overviewAdvantages of the Silencer siRNA Cocktail Kit approach to making siRNA

B. Reagents Provided with the Kit and StorageC. Materials Not Provided with the KitGene-specific template(s)For dsRNA purification:To assess the reaction products:


II. Preparation of Template DNAA. Choosing the Target SiteB. Strategies for Transcription of dsRNAFigure 1. T7 Polymerase Promoter: Minimal Sequence Requirement

C. PCR Templates1. Amplification strategies to add T7 promoter sequences to DNAFigure 2. Adding T7 promoters by PCR

2. PCR amplification profile suggestionsFigure 3. Strategy for Adding a Single T7 Promoter by PCR

3. Check PCR products on a gel before using them in this procedure4. Purification of the PCR products

D. Plasmid Templates1. Cloning strategyFigure 4. Cloning in plasmids

2. Plasmid linearizationFigure 5. Linearized plasmids

3. After linearization4. Plasmid DNA purity

III. Silencer siRNA Cocktail Kit ProtocolA. Before Using the Kit for the First TimePrepare the Wash Solution

B. Transcription Reaction Assembly1. Thaw the frozen reagents at room temp and place them in ice2. Assemble transcription reaction at room temperature3. Mix thoroughly4. Incubate at 37˚C for 2 hr

C. Annealing RNA to Maximize Duplex Yield1. Mix the transcription reactions containing complementary RNA2. Incubate at 75˚ for 5 min, then cool to room temperature3. (optional) Check 1/400th of the dsRNA on an agarose gel

D. Nuclease Digestion to Remove DNA and ssRNA1. Assemble RNase A digestion reaction on ice2. Incubate at 37˚C for 1 hr

E. Purification of dsRNA1. Add 10X Binding Buffer and 100% ethanol to the dsRNA2. Apply dsRNA mixture to the Transcription Reaction Filter Cartridge, and draw it through3. Wash the Transcription Reaction Filter Cartridge with 2 X 500 µl Wash Solution4. Recover the dsRNA with two serial elutions in 50 µl hot Elution Solution5. Quantitation and storage of the dsRNA6. (optional) Check 1/400th of the purified dsRNA on an agarose gel

F. RNase III Digestion and siRNA Purification1. Assemble RNase III digestion reaction in a microfuge tube2. Incubate 1 hr at 37˚C3. Prewet the siRNA Purification Unit, then purify the siRNA by spinning it through4. Determine the concentration of the siRNA5. (optional) Check the siRNA on a 15% acrylamide gel

G. siRNA Cocktail Quantification1. Measure the A260 of a 1:25 dilution of the siRNA2. Determine the concentration of the siRNA cocktail in µg/ml3. Determine the molar concentration of the siRNA cocktail4. Example calculation

H. Transfecting Mammalian Cells

IV. TroubleshootingA. Use of the GAPDH Control Template1. Positive control reaction instructions2. What to do if the positive control reaction doesn’t work as expectedFigure 6. Positive Control Reaction

B. Troubleshooting Low Yield from the Transcription Reaction1. Neither my template nor the control reaction works2. The control reaction works, but my template gives low yieldFigure 7. Possible outcomes of mixing experiment

C. Troubleshooting Unexpected Transcription Reaction Products1. Transcription reaction products produce a smear when run on a gel2. Transcription reaction products run as more than one band, or as a single band smaller than expected3. Transcription reaction products are larger than expected

D. Troubleshooting RNase III Digestion and siRNA Purification1. My dsRNA is not digested by RNase III2. My RNA concentration is drastically reduced after purification on an siRNA Purification Unit

V. Additional ProceduresA. Quantitation of RNA by SpectrophotometryB. Agarose and Acrylamide Gel Electrophoresis Instructions1. Solutions for gel electrophoresis2. Pouring and running agarose gels3. Pouring and running acrylamide gels

VI. AppendixA. ReferencesB. Silencer siRNA Cocktail Kit SpecificationsKit contents and storage:Stability:Quality Control


silencer™ sirna cocktail kit (rnase iii) · silencer™ transfection kit cat #1630 the silencer...

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