Thermo Fisher Scientific Silencer® siRNA Cocktail Kit User manual

Brand: Thermo Fisher Scientific

Model: Silencer® siRNA Cocktail Kit

Type: User manual

Silencer

siRNA Cocktail Kit (RNase III)

(Cat #AM1625)

Instruction Manual

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A. Background

B. Reagents Provided with the Kit and Storage Conditions

C. Materials Not Provided with the Kit

D. Related Products Available from Ambion

II. Preparation of Template DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A. Choosing the Target Site

B. Strategies for Transcription of dsRNA

C. PCR Templates

D. Plasmid Templates

III.

Silencer

siRNA Cocktail Kit Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

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 Quantitation

H. Transfecting Mammalian Cells

IV. Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

A. Use of the Positive Control Template

B. Low Yield from the Transcription Reaction

C. Unexpected Transcription Reaction Products

D. Troubleshooting RNase III Digestion and siRNA Puriﬁcation

V. A d d i t i o n a l Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

A. Quantitation of RNA by Spectrophotometry

B. Agarose and Acrylamide Gel Electrophoresis Instructions

C. Miniprep for Isolating Transcription-quality Plasmid DNA

VI. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

A. References

Silencer

siRNA Cocktail Kit Speciﬁcations

C. Quality Control

Manual 1625M Revision B Revision Date: February 16, 2007

For research use only. Not for use in diagnostic procedures. By use of this product, you accept the terms and

conditions of all applicable Limited Label Licenses. For statement(s) and/or disclaimer(s) applicable to this

product, see below.

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.

Warranty and Liability

Ambion is committed to providing the highest quality reagents at competitive prices. Ambion

warrants that for the earlier of (i) one (1) year from the date of shipment or (ii) until the shelf life date, expiration date, “use

by” date, “guaranty date”, or other end-of-recommended-use date stated on the product label or in product literature that

accompanies shipment of the product, Ambion's products meet or exceed the performance standards described in the prod-

uct specification sheets if stored and used properly. No other warranties of any kind, expressed or implied, are provided by

Ambion. WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE ARE

EXPRESSLY DISCLAIMED. AMBION'S LIABILITY SHALL NOT EXCEED THE PURCHASE PRICE OF THE

PRODUCT. AMBION SHALL HAVE NO LIABILITY FOR INDIRECT, CONSEQUENTIAL, OR INCIDENTAL

DAMAGES ARISING FROM THE USE, RESULTS OF USE, OR INABILITY TO USE ITS PRODUCTS. See the full

limited warranty statement that accompanies products for full terms, conditions and limitations of Ambion's limited prod-

uct warranty, or contact Ambion for a copy.

Patents and Licensing Notiﬁcations

The

Silencer

siRNA Cocktail Kit (RNase III) is covered by US patents and for-

eign patents pending. Ambion has been granted rights by the Massachusetts Institute of Technology to US Patent Applica-

tions 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 compo-

nents 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.

This product is licensed under European Patents 1144623, 1214945 and foreign equivalents from Alnylam Pharmaceuticals,

Inc., Cambridge, USA and is provided only for use in academic and commercial research including in vitro and in vivo iden-

tification and validation of drug targets (but excluding the evaluation or characterization of this product as the potential basis

for a siRNA-based drug) and not for any other commercial purposes. Information about licenses for commercial use (includ-

ing discovery and development of siRNA-based drugs) is available from Alnylam Pharmaceuticals, Inc., 300 Third Street,

Cambridge, MA 02142, USA.

Ambion/AB Trademarks

Applied Biosystems, AB (Design), and Applera are registered trademarks of Applera

Corporation or its subsidiaries in the US and/or certain other countries. Ambion, The RNA Company,

Silencer

, MEGAscript and RNase

Zap

are registered trademarks of Ambion, Inc. in the U.S. and/or certain

other countries. All other trademarks are the sole property of their respective owners.

I.A. Background

Introduction

I. Introduction

A. Background

The

Silencer

siRNA Cocktail Kit (RNase III) 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 tran-

scription 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 2003). Using this method, a large region of the mRNA of inter-

est can be targeted for RNAi at once.

RNA interference and short

interfering RNAs

RNA interference (RNAi), the phenomenon by which dsRNA specifi-

cally suppresses expression of a target gene, was originally discovered in

worms (Fire 1998), but has now been found in a large number of organ-

isms, including flies (Misquitta and Paterson 1999), trypanosomes

(Ngo 1998), planaria (Sánchez-Alvarado and Newmark 1999), hydra

(Lohmann 1999), and zebrafish (Wargelius 1999). RNAi is becoming

one of the most widely used methods for studying gene function in

mammalian cells.

In the RNAi pathway in vivo, long double-stranded RNA (dsRNA)

introduced into a cell is cleaved into a mixture of short dsRNA mole-

cules called short interfering RNA (siRNA). The enzyme that catalyzes

the cleavage, Dicer, is an endo-RNase that contains RNase III domains.

The siRNAs produced by Dicer are 21–23 bp in length and contain

3' dinucleotide overhangs with 5'-phosphate and 3'-hydroxyl termini

(Bernstein 2001, Elbashir 2001, Grishok 2001, Hamilton and Baul-

combe 1999, Knight and Bass 2001, and Zamore 2000). siRNAs asso-

ciate with a cellular complex, called the RNA-induced silencing

complex (RISC), and guide the complex to their target mRNA through

base pairing interactions (Hammond 2001). Nuclease components of

the RISC then cleave the target mRNA (Tuschl 1999, Zamore 2000),

causing a reduction in target gene expression.

RISC

RNase

21–23 bp

siRNAs

siRNAs associate

with RISC complex

Cleavage of mRNA

by RISC

dsRNA cleavage

Degradation by

exonucleases

long dsRNA

RNA Interference in vivo

Silencer

siRNA Cocktail Kit (RNase III)

I.A. Background

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 reac-

tion to synthesize two complementary RNA transcripts from templates

supplied by the user. Next, the transcription template(s) and any sin-

gle-stranded RNA (ssRNA) are removed by nuclease digestion, and the

long dsRNA is purified with a solid-phase adsorption system (Tran-

scription Reaction Filter Cartridges).

The long dsRNA is then cleaved with RNase III in a 1 hour reaction to

produce siRNAs. The recombinant

E. coli

RNase III used for this reac-

tion cleaves long dsRNA into dsRNAs containing a 5'-phosphate

3'-hydroxyl, and a dinucleotide 3' overhang, the same structure as siR-

NAs produced in vivo. The reaction products are heterogeneous in

length, ranging from 12-30 bp, with the majority of the reaction prod-

ucts being 12-15 bp in length.

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 specif-

ically inducing RNAi in mammalian cells (Yang 2002, Calegari 2002,

Byrom 2003, Trotta, 2003).

Advantages of the

Silencer

siRNA Cocktail Kit approach

to making siRNA

Methods for producing siRNA include chemical synthesis, in vitro tran-

scription (e.g., using this kit, or Ambion’s

Silencer

siRNA Construction

Kit), and using siRNA expression vectors. The

Silencer

siRNA Cocktail

Kit approach eliminates the need to screen several different siRNAs 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. We find that only about one out of four

randomly chosen siRNA sequences can reduce the expression of the tar-

get by 80% or more. Thus, synthesizing a 150 bp dsRNA and digesting

it into an siRNA cocktail using this kit raises the chance that the mix-

ture will contain a functional siRNA to nearly 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 cul-

ture plates.

Nuclease Digestion (37˚C)

1 hr

RNase III Digestion (37˚C)

1 hr

dsRNA Purification

10–15 min

siRNA Purification

10 min

Transfection

Transcription

2– 4 hr

5' 3'

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

Introduction

B. Reagents Provided with the Kit and Storage Conditions

The kit contains components to synthesize 20 dsRNAs and to digest a

total of 600 µg of dsRNA into siRNA cocktails.

Amount Component Storage

1.75 mL Nuclease-free Water any temp*

*Store Nuclease-free Water at –20°C, 4°C or room temp

20 Transcription Rxn Filter Cartridges room temp

60 Collection Tubes room temp

20 siRNA Purification Units room temp

12 mL 2X Wash Solution

Add 12 mL 100% ethanol before use

–20°C

40 µL T7 Enzyme Mix –20°C

40 µL 10X T7 Reaction Buffer –20°C

4mL Elution Solution –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

40 µL RNase A –20°C

600 µL RNase III (1 U/µL) –20°C

500 µL 10X RNase III Buffer –20°C

45 µL DNase I (2 U/µL) –20°C

100 µL 10X Digestion Buffer –20°C

10 µL GAPDH Control Template (250 ng/µL) –20°C

Silencer

siRNA Cocktail Kit (RNase III)

I.C. Materials Not Provided with the Kit

C. Materials Not Provided with the Kit

Gene-specific template(s)

A transcription template(s) is needed with T7 RNA polymerase pro-

moters positioned to transcribe sense and antisense RNA corresponding

to the target RNA. See section II starting on page 6 for a detailed discus-

sion 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

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

siRNA Transfection II

Kit

Cat #AM1631

The

Silencer

siRNA Transfection II Kit contains both siPORT™

NeoFX™

and siPORT

Lipid

Transfection Agents in addition to a well-characterized

siRNA targeting human, mouse, and rat GAPDH. This kit is ideal for devel-

oping an optimal transfection protocol for your cells. Also included are a

highly validated non-targeting negative control siRNA and a detailed

Instruction Manual.

RNase III

Cat #AM2290

Escherichia coli

Ribonuclease III (RNase III; EC 3.1.24) is a double-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 prod-

ucts 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).

Silencer

siRNA Labeling Kits

Cat #AM1632, AM1634

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.

Silencer

siRNA Controls

Cat #AM4250–AM4639

see our web or print catalog

www.ambion.com/siRNA

Silencer

siRNA Controls are chemically synthesized siRNAs for genes com-

monly used as controls. Validated control siRNAs are available for genes such as

GAPDH,

-actin, cyclophilin, KIF11 (Eg5), GFP, and luciferase. These siR-

NAs are ideal for developing and optimizing siRNA experiments and have been

validated for use in human cells; many are also validated in mouse and rat cells.

RNase

Zap

Cat #AM9780, AM9782, AM9784

RNase Decontamination Solution. RNase

Zap

is simply sprayed, poured, or

wiped onto surfaces to instantly inactivate RNases. Rinsing twice with dis-

tilled water will eliminate all traces of RNase and RNase

Zap

I.D. Related Products Available from Ambion

Introduction

Electrophoresis Reagents

see our web or print catalog

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 website

(www.ambion.com) for a complete listing as this product line is always growing.

Antibodies for siRNA Research

see our web or print catalog

For select

Silencer

Control and Validated siRNAs, Ambion offers corre-

sponding antibodies for protein detection. These antibodies are ideal for con-

firming mRNA knockdown results by analyzing concomitant protein levels.

Silencer

siRNA Cocktail Kit (RNase III)

II.A. Choosing the Target Site

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 differ-

ent parts of mRNA (i.e. 3' end, 5' end or the center of the message); siRNA

cocktails targeting any part of an mRNA should be effective 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 populations

for gene silencing experiments (Yang 2002, Calegari 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 cocktail with fewer unique siRNAs, but each

one is at a higher concentration than when a larger dsRNA substrate is

digested. A 200 bp dsRNA is typically digested into 7–16 different

siRNAs. Since one in four randomly chosen siRNAs generally have

RNAi activity when introduced into cells, 150–500 bp dsRNAs are very

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 can both be added to a single reaction.

Although both templates should be transcribed at the same rate, if

they are not, the final dsRNA yield will be dictated by the template

with the lower transcription efficiency. Alternatively, the two tem-

plates can be transcribed in separate reactions to make complemen-

tary RNA molecules which are then mixed and annealed. For the

annealing step, the two transcripts can be mixed in precisely equimo-

II.C. PCR Templates

Preparation of Template DNA

lar amounts. 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 (Figure 2);

this generates templates that can be directly added to the

Silencer

siRNA

Cocktail Kit transcription reaction. Begin by synthesizing PCR primers

with the T7 promoter sequence appended to the 5' end of the primer

(see Figure 2 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 the annealing temperatures of the entire PCR primer (with

the T7 promoter site) and the gene specific portion of the PCR

primer separately.

•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

for

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'

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

•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 when 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 transcrip-

tion reaction.

T7 gene specific

gene specific gene specific

gene specific T7 gene specific

gene specific

Silencer

siRNA Cocktail Kit (RNase III)

II.C. PCR Templates

the gene-specific region of the primer. We have found that using the

calculated annealing temperature for the initial cycles often results in

synthesis of spurious PCR products.

•Once some PCR product is made, subsequent primer annealing

events (cycle 6 and thereafter) use the entire primer site; therefore use

the calculated T

for the

entire

PCR primer plus ~5°C for subse-

quent cycles.

•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 purification tech-

niques. Note that optimizing the PCR conditions may result in ampli-

fication of a single band.

Figure 2. Strategy for Adding a Single T7 Promoter by PCR

PCR Primer

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

PCR

Trxn

RNA Transcript

II.D. Plasmid Templates

Preparation of Template DNA

D. Plasmid Templates

1. Cloning strategy When using plasmid templates in the Silencer siRNA Cocktail Kit, it is best

to make two separate clones with the same target region in both orienta-

tions.

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 orien-

tations with regard to the T7 promoter.

•Or, include the T7 promoter sequence at the 5' end of one or both of

the PCR primers, then perform PCR to incorporate them into the

fragment (see Figure 2 on page 8). Finally, ligate the PCR product

into a PCR cloning vector (one that does not include a T7 pro-

moter).

•Or, 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. Ligate the PCR product into a cloning vector using the

added 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 template, then exam-

ine the DNA on a gel to confirm that cleavage is complete. Since initia-

tion of transcription is the rate limiting step 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 syn-

thetic capacity of the reaction.

Figure 3. Cloning in plasmids

T 7 Promoter T 7 Promote

NOTE

We routinely use all types of restric-

tion enzymes. However, there has

been one report of low level tran-

scription from the inappropriate tem-

plate strand in plasmids cut with

restriction enzymes leaving 3' over-

hanging ends (produced by Kpn I,

Pst I, etc.; Schenborn and Mierindorf,

1985).

Figure 4. Linearized plasmids

T 7

Silencer® siRNA Cocktail Kit (RNase III)

II.D. Plasmid Templates10

3. After linearization Terminate the restriction digest by adding each of the following:

•1/20 volume 0.5 M EDTA

•1/10 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,

respin 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. The

greatest yields of dsRNA will be obtained with very clean template prep-

arations. 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 before performing the transcription reaction

(section IV. Proteinase K treatment on page 21).

III.A. Before Using the Kit for the First Time

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 then 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.

Microcentrifuge 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

Assemble the transcription reaction at room temperature in the order

shown below. The following amounts are for a single 20 µL transcrip-

tion reaction. Reactions may be scaled up or down if desired.

Amount Component

to 20 µL Nuclease-free Water

0.2– 2 µg Linear template DNA*

*Templates <500 bp: Use 200–500 ng of template with opposing T7 promoters, or

use 200–500 ng of each template when the T7 promoter template is on separate

molecules.

Templates ≥500 bp: Use 1 µg of a template with opposing T7 promoters, or use

1 µg of each template when the T7 promoter template is on separate molecules.

2µL 10X T7 Reaction Buffer

2µL ATP Solution

2µL CTP Solution

2µL GTP Solution

2µL UTP Solution

2µL T7 Enzyme Mix

Silencer® siRNA Cocktail Kit (RNase III)

III.C. Annealing RNA to Maximize Duplex Yield12

3. Mix thoroughly Gently flick the tube or pipette the mixture up and down, then briefly

microcentrifuge 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

Include this annealing step for the following types of reactions:

•for all >800 nt dsRNA synthesis reactions

•for ≤800 nt dsRNA synthesis reactions when the two strands were

synthesized from separate transcription templates (in the same or in

separate transcription reactions).

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 struc-

tures) rather than the dsRNA.

1. Mix the transcription

reactions containing

complementary RNA

•If sense and antisense RNA were synthesized in separate transcrip-

tion reactions, simply add the entire contents of one of the reactions

to the other.

If desired, reserve a 0.5 µL aliquot of each template before mixing for

gel analysis.

•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.

2. Incubate at 75°C 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.

Do not put the reaction on ice to cool.

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.

•Dilute the gel samples in TE (10 mM Tris, 1 mM EDTA) or in gel

loading buffer.

(Instructions for running a gel are in section V.B.2 on page 26.) The

dsRNA will migrate slightly slower than DNA markers of the same

length.

III.D. Nuclease Digestion to Remove DNA and ssRNA

Silencer siRNA Cocktail Kit Protocol

D. Nuclease Digestion to Remove DNA and ssRNA

This DNase/RNase A treatment digests template DNA and any ssRNA

that did not anneal. RNase A will not degrade dsRNA when using the

reaction conditions specified below.

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.

E. Purification of dsRNA

This purification removes proteins, free nucleotides, and nucleic acid

degradation products from the dsRNA.

NOTE

For the quickest dsRNA purification, preheat the Elution Solution to ~95°C

before starting the purification procedure.

1. Assemble the dsRNA

binding mix

Assemble the dsRNA binding mix by adding 10X Binding Buffer,

water, and 100% ethanol to the dsRNA according to the table below.

Gently mix the reaction by pipetting up and down.

Amount Component

20 µL dsRNA (from step B.4 or step C.2)

21 µL Nuclease-free Water

5µL 10X Digestion Buffer

2µL DNase I

2µL RNase A

Amount Component

50 µL dsRNA (from step D.2 above)

50 µL 10X Binding Buffer

150 µL Nuclease-free Water

250 µL 100% Ethanol

Silencer® siRNA Cocktail Kit (RNase III)

III.E. Purification of dsRNA14

2. Apply binding mix to the

Transcription Reaction

Filter Cartridge, and draw

it through

Pipet the entire 500 µL dsRNA binding mix onto the filter in the Tran-

scription Reaction Filter Cartridge, and draw it through by centrifuga-

tion 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.

3. Wash the Transcription

Reaction Filter Cartridge

with 2 X 500 µL Wash

Solution

IMPORTANT

Verify that 12 mL of 100% ethanol was added to the 2X Wash Solution.

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 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.

III.E. Purification of dsRNA

Silencer siRNA Cocktail Kit Protocol

4. Recover the dsRNA

2 X 50 µL Elution Solution

NOTE

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 solu-

tion (≤30 mM).

a. Transfer the 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 elu-

tion is included to recover any remaining RNA.

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 25).

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 26.) The

dsRNA will migrate slightly slower than DNA markers of the same

length.

Silencer® siRNA Cocktail Kit (RNase III)

III.F. RNase III Digestion and siRNA Purification16

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. Digesting 15 µg of long

dsRNA will yield enough siRNA for about 120 transfections in wells of

a 24-well plate at 50 nM final concentration.

1. Assemble RNase III

digestion reaction in a

microcentrifuge 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

draw the siRNA 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 digestion 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 25).

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 26 for

instructions). See Figure 5 on page 20 for an example of how the siRNA

will look on a gel.

Amount Component

up to 15 µg dsRNA (up to 30 µL)*

*If your long dsRNA concentration is <0.5 µg/µL, you have two choices:

1) Digest <15 µg 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 accommodate 15 µg of your long dsRNA.

15 µL RNase III

5µL 10X RNase III Buffer

to 50 µL Nuclease-free Water

III.G. siRNA Cocktail Quantitation

Silencer siRNA Cocktail Kit Protocol

G. siRNA Cocktail Quantitation

The concentration of the siRNA cocktail 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. 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 spectro-

photometer. Be sure to blank the spectrophotometer 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 1000 to determine the concentra-

tion 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 deter-

mined by dividing the µg/mL concentration of the siRNA by 9 (expla-

nation below).

•There are 9 µg of RNA in 1 nmol of a 13.5 bp dsRNA:

•Dividing the µg/mL concentration by 9 yields the µM concentration

as shown below:

4. Example calculation A 1:25 dilution of purified siRNA has an A260 = 0.4. The molar concen-

tration is determined as follows:

1000 = 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

nmol

9 µg =X µg

mL Xnmol

9 µg =mL (9)

X nmol =X µmol

L (9) =X µM

Therefore µM = X ÷ 9

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

Silencer® siRNA Cocktail Kit (RNase III)

III.H. Transfecting Mammalian Cells18

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 50–100 nM; however, a more extensive concentra-

tion 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 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, add-

ing fresh medium does not appear to have a detrimental effect on the

activity of the transfected siRNAs.

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Thermo Fisher Scientific Silencer® siRNA Cocktail Kit User manual

Brand: Thermo Fisher Scientific

Model: Silencer® siRNA Cocktail Kit

Type: User manual

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Thermo Fisher Scientific Silencer® siRNA Cocktail Kit User manual

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Thermo Fisher Scientific Silencer® siRNA Cocktail Kit User manual

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