Mission Sirna - Better Sirna Design, Better Rnai Performance

Better siRNA Design, Better RNAi Performance

X79713-siRNA brochure v2.indd cov1

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Introduction to RNAi

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Pre-designed siRNA for Human, Rat, and Mouse

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Pre-arrayed siRNA Libraries

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Custom siRNA Synthesis

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siRNA Transfection

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RNA Isolation and Purification

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Knockdown Detection– mRNA Level

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High-Throughput Knockdown Detection–mRNA Level

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Knockdown Detection– Protein Level

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Sigma siRNA Workflow Products in Action

9/19/07 9:59:51 AM

Introduction to RNAi RNA interference (RNAi) is a natural biological mechanism wherein small interfering RNA (siRNA) duplexes induce potent inhibition of gene expression. These siRNA duplexes are produced naturally when an enzyme, Dicer, cleaves long double-stranded RNA (dsRNA). Alternatively, synthetic versions of siRNA can be introduced into the cell, thus triggering the remainder of the RNAi pathway. The resulting 21-23 nucleotide fragments, termed siRNA, associate with an RNase-containing complex to form the RNA-induced

silencing complex (RISC), which unwinds the siRNA duplex and releases the sense strand. The RISC-bound antisense strand then serves as a guide for targeting the activated complex to complementary mRNA sequences, resulting in subsequent mRNA cleavage and degradation. In effect, only catalytic amounts of siRNA are required for destruction of mRNA, resulting in the knockdown or silencing of the target gene and diminished protein expression.

RNA Interference with MISSION siRNA Processing by Dicer

Sigma siRNA Workflow

Select Target Gene

Order siRNA Duplexes

Deliver siRNA to Cells

Detect Knockdown

Pre-designed MISSION® siRNA MISSION siRNA Libraries Custom siRNA Synthesis

N-TER™ siRNA Transfection System

GenElute™ Mammalian Total RNA Miniprep Kits Quantitative RT-PCR QuantiGene® 2.0 RNA Quantification System Sigma Antibodies

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Pre-designed siRNA for Human, Rat, and Mouse MISSION siRNA Current studies suggest that the nucleotide sequence of an siRNA is an extremely important, if not the most important, factor for a successful RNAi experiment. Understanding this requirement, Sigma-Aldrich has entered into an exclusive partnership with Rosetta Inpharmatics, a leader in advanced siRNA research, to use the proprietary Rosetta siRNA Design Algorithm to design its MISSION line of siRNA. The Rosetta siRNA Design Algorithm utilizes Position-Specific Scoring Matrices (PSSM) and knowledge of the all-important siRNA seed region1 to predict the most effective and specific siRNA sequences for your target gene of interest. Additionally, the Rosetta siRNA Design Algorithm has been trained with feedback from over 3 years of gene-silencing experiments, ensuring that the algorithm’s in silico rules are guided and bolstered by realworld empirical evidence.

Using a siRNA designed with a best-in-class algorithm saves time and money, enabling you to focus on downstream applications, not up-front siRNA design and validation work.

Benefits of pre-designed MISSION siRNA, powered by the Rosetta siRNA Design Algorithm: ■ ■ ■ ■ ■

Increased target specificity, due to siRNA seed region optimization rules1 Efficient knockdown of low abundance messages Guaranteed gene silencing High quality siRNA produced at ISO 9000:2001 certified sites Freedom to operate for research use The MISSION siRNA Performance Guarantee Sigma guarantees that 2 out of 3 siRNA duplexes per target gene will achieve knockdown efficiencies of greater than or equal to 75%.

100

% Expression Remaining

90 80 70 60 50 40 30 20 10 ST 7 TA B3 TM 4S F1

SLK SM U1 SM UR F2

RE LA RIO K1 RN F1 SLC 0 22 A3

PB K PC NT 1 PD P2 PM E-1 PP M1 A PP P1 R1 1 PP P2 R1 A PT PL A RA B2 2A

NL N NR 2F 2

MB IP MB TP S2 ME LK MS T4 MT MR 2 NE K7

Co ntr ol CC ND 1 CR K7 DD X4 1 DD X4 8 EG LN 1 GL TS CR 2 HIP K1 HR PT 2 IHP K2 IRA LO K4 C1 23 16 MA P2 K2 MA ST L

0

Silencing efficacy of representative MISSION siRNAs designed using the Rosetta siRNA Design Algorithm. Target mRNA levels were measured by QuantiGene® Reagent System from samples harvested 24 hours after transfection into HeLa cells.

Selected References 1. Jackson, A.L., et al., Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA, 12, 1179-1187 (2006) 2. Jackson, A.L., et al., Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol., 21, 635-637 (2003) 3. Majercak, J., et al., LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer’s disease. Proc. Natl. Acad. Sci. USA. Nov 21;103(47):17967-72. Epub 2006 Nov 10. PMID: 17098871 [PubMed -indexed for MEDLINE] (2006)

Ordering Information We’ve made ordering MISSION siRNA for single gene targets easy through Sigma’s state-of-the-art Web interface, Your Favorite Gene at sigma.com/yfg. Simply search against your gene of interest by gene name, symbol, RefSeq, or Gene ID number. For each gene search you can order the MISSION siRNA available for that particular gene.

4. Espeseth, A.S., et al., A genome wide analysis of ubiquitin ligases in APP processing identifies a novel regulator of BACE1 mRNA levels. Mol. Cell Neurosci. Nov;33(3):227-35. Epub 2006 Sep 15. (2006)

Our Innovation, Your Research

X79713-siRNA brochure v2.indd 1

Your Favorite Gene

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TM

Shaping the Future of Life Science

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Pre-arrayed siRNA Libraries MISSION siRNA Libraries

Benefits of MISSION siRNA Libraries:

MISSION siRNA Libraries offer the most compelling siRNA collections for high-throughput screening by targeting genes of high therapeutic value as defined with input from major pharmaceutical companies. The flexible format of MISSION siRNA libraries facilitates research for life scientists who are interested in specific classes of genes as well as those who need to generate information across the entire druggable genome.

■

siRNA designed using the powerful Rosetta siRNA Design Algorithm, providing for more efficient gene knockdown and greater target specificity ■ 3 individual siRNA provided per gene, allowing for optional siRNA pooling steps ■ siRNA provided at quantities to allow for multiple screenings and hit follow-up ■ Gene targets picked in collaboration with major pharmaceutical companies, and based on latest NCBI classifications Product Specifications 21-mer siRNA duplexes; 19 bp of target sequence with 3’ dTdT overhangs 3 individual siRNA duplexes per target gene All siRNA duplexes spotted in 96-well microplates with 80 duplexes per plate. First and last columns of each plate are empty

Ordering Information

Positive control siRNA sequences included on all plates

MISSION siRNA Human Libraries

2

Cat. No.

Product Name

SI00100-1SET

MISSION siRNA Human Druggable Genome Library

SI01100-1SET SI02100-1SET SI03100-1SET

MISSION siRNA Rat Libraries Targets

Qty.

6650

1 nmol

Cat. No. SI20100-1SET

MISSION siRNA Human Ligase Panel

949

1 nmol

SI21100-1SET

MISSION siRNA Human Kinase Panel

714

1 nmol

SI22100-1SET

MISSION siRNA Human Phosphatase Panel

293

1 nmol

SI04100-1SET

MISSION siRNA Human Growth Factors and Receptors Panel

375

1 nmol

SI05100-1SET

MISSION siRNA Human Cell Adhesion and Cytoskeleton Panel

496

1 nmol

SI06100-1SET

MISSION siRNA Human Ion Channel and Transporters Panel

639

1 nmol

SI07100-1SET

MISSION siRNA Human Assorted Function Panel

228

1 nmol

SI08100-1SET

MISSION siRNA Human GPCR Panel

304

1 nmol

SI09100-1SET

MISSION siRNA Human Hydrolase Panel

204

SI10100-1SET

MISSION siRNA Human Metabolism and Cell Traffic Panel

SI11100-1SET SI12100-1SET

Targets 6025

1 nmol

MISSION siRNA Rat Ligase Panel

816

1 nmol

MISSION siRNA Rat Kinase Panel

669

1 nmol

SI23100-1SET

MISSION siRNA Rat Phosphatase Panel 266

1 nmol

SI24100-1SET

MISSION siRNA Rat Growth Factors and Receptors Panel

342

1 nmol

SI25100-1SET

MISSION siRNA Rat Cell Adhesion and Cytoskeleton Panel

433

1 nmol

SI26100-1SET

MISSION siRNA Rat Ion Channel and Transporters Panel

594

1 nmol

SI27100-1SET

MISSION siRNA Rat Assorted Function Panel

210

1 nmol

SI28100-1SET

MISSION siRNA Rat GPCR Panel

275

1 nmol

SI29100-1SET

MISSION siRNA Rat Hydrolase Panel

188

1 nmol

1 nmol

SI30100-1SET

MISSION siRNA Rat Metabolism and Cell Traffic Panel

201

1 nmol

217

1 nmol

SI31100-1SET

MISSION siRNA Rat Nucleic Acid Binding Panel

287

1 nmol

MISSION siRNA Human Nucleic Acid Binding Panel

310

1 nmol

SI32100-1SET

MISSION siRNA Rat Oxidoreductase Panel

302

1 nmol

MISSION siRNA Human Oxidoreductase Panel

338

1 nmol

SI33100-1SET

MISSION siRNA Rat Protease Panel

341

1 nmol

SI13100-1SET

MISSION siRNA Human Protease Panel

379

1 nmol

SI34100-1SET

MISSION siRNA Rat Cell Surface and Nuclear Receptors Panel

647

1 nmol

SI14100-1SET

MISSION siRNA Human Cell Surface and Nuclear Receptors Panel

722

1 nmol

SI35100-1SET

MISSION siRNA Rat Cell Regulation Panel

214

1 nmol

SI15100-1SET

MISSION siRNA Human Cell Regulation Panel

227

1 nmol

SI36100-1SET

MISSION siRNA Rat Transfer and Carrier Proteins Panel

66

1 nmol

SI16100-1SET

MISSION siRNA Human Transfer and Carrier Proteins Panel

71

1 nmol

SI37100-1SET

MISSION siRNA Rat Transferase Panel

174

1 nmol

SI17100-1SET

MISSION siRNA Human Transferase Panel

184

1 nmol

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Product Name MISSION siRNA Rat Druggable Genome Library

Qty.

MISSION siRNA Mouse Libraries Cat. No. SI42050-1SET

Product Name

Targets Qty. 623 500 pmol

MISSION siRNA Mouse Kinase Panel

TECHNICAL SERVICE: 800-325-5832

9/19/07 9:59:57 AM

Custom siRNA Synthesis siRNA Synthesis According to Your Specification Don’t see a pre-designed MISSION siRNA that will work for you? Scientists at Sigma have identified key factors related to the synthesis of siRNA that are important for effective knockdown and have developed an RNA synthesis platform that delivers consistent high-quality siRNA. Our RNA synthesis relies on the use of fast deprotection ribonucleoside phosphoramidites protected at the 2’ position by a tert-butyldimethylsilyl (TBDMS) group and, at the 3’ position, by a phosphoramidite. Optimized, proprietary processes built around this unique platform allow for higher coupling efficiency and faster deprotection, resulting in higher quality siRNA synthesis and faster turnaround times.

Benefits of Sigma’s Custom siRNA Synthesis Service: ■

High-quality and cost-effective siRNA synthesis ■ Fast turn-around times ■ Reduced need for costly and time-consuming purifications ■ High-throughput capacity for large projects

PAGE

RP-HPLC

Yields Guaranteed yield (OD)

2

5

10

50

1

5

10

50

Approx. yield (nmols*)

10

25

50

250

5

25

50

250

750 wells

3750 wells

75 wells

375 wells

750 wells

3750 wells

Transfections (approx.) 24-well plate format

150 wells

375 wells

Please inquire for alternative quantities, purification grades and labels.

Turn-Around Time for siRNA Oligos Desalted

PAGE

RP-HPLC

Yields Guaranteed yield (OD)

2

5

10

50

1

5

10

50

Approx. yield (nmols*)

10

25

50

250

5

25

50

250

4

5

5

6

8

8

8

5

6

6

7

9

9

9

6

6

7

9

9

9

7

7

8

10

10

10

Turn-around time (TAT) Single-strand, unlabeled No. of days

4

Ordering Information Those wishing to have siRNA synthesized according to their own design and specifications may do so at: sigma.com/custom_sirna.

Guaranteed Yields and Transfections for siRNA Oligos Desalted

Sigma’s siRNA is produced at sites around the world, providing for first-class turn-around times and customer service.

Simply use the Sigma siRNA configuration tools to supply us with: ■ Your sequence to be synthesized ■ Amount to be synthesized ■ siRNA Purification Level desired: • Standard Desalted • HPLC purification • PAGE purification ■ Number of tubes to aliquot into ■ Modifications desired: • 5’ and 3’ Labels – 6-FAM – Amine – Biotin – Cy®3 – Cy5 – Cy5.5 – Fluorescein – Phosphate • Internal Modifications – 2’ O-Methyl RNA – LNA

Single-strand, labeled No. of days

5

Guaranteed duplex, unlabeled No. of days

5

5

Guaranteed duplex, labeled No. of days

6

6

Note: Turn-around time is dependent upon successful QC validation, and does not include delivery time. Please check with your local sales representative for local turn-around times. *Estimate 1 OD = 5 nmols = 30 µg for a 20 mer oligo

Our Innovation, Your Research

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siRNA Transfection N-TER™ Nanoparticle siRNA Transfection System

Sigma’s N-TER Nanoparticle siRNA Transfection System is based on a peptide transfection reagent specifically designed to bypass these limitations and allow for efficient delivery of siRNAs into these historically recalcitrant eukaryotic cell types.

Knockdown of GAPDH in Human Astrocyte

Primary Cells

100

% GAPDH Expression

Traditional lipid-based siRNA transfection reagents exhibit a number of drawbacks, including a limited ability to transfect into a variety of cell types, such as primary, neuronal, differentiated, and nondividing cells.

80 60 40 20 0 20 nM

Benefits of the N-TER Nanoparticle siRNA Transfection System: ■

Efficient transfection of a wide variety of historically hard-totransfect cells, including differentiated and non-dividing cells ■ Stocks of N-TER/siRNA Nanoparticles can be stored for at least 1 year at –20 °C and used for subsequent transfections, increasing standardization and reproducibility in all transfection experiments targeting the same gene ■ Fast and simple protocol easily adapted for high throughput and reverse transfection applications For more information, visit us online at sigma.com/nter.

N-TER has been validated to work in these cell types:

4

3T3-L1, differentiated Mouse, embryonic fibroblast cell line

HEK293T Human, embryonic kidney cell line

MDA-MB-231 Human, breast adenocarcinoma cell line

A2780 Human, ovarian carcinoma cell line

HeLa Human, cervical adenocarcinoma cell line

NHA Human, astrocyte primary cell

A431 Human, ovarian carcinoma cell line

Hepatocyte Rat, hepatocyte primary cell

NHEK-AD Human, adult keratinacyte primary cell

A549 Human, lung carcinoma cell line

HepG2 Human, hepatocarcinoma cell line

RAW264.7 Mouse, macrophage cell line

ASPC-1 Human, pancreatic carcinoma cell line

HT-29 Human, colorectal adenocarcinoma cell line

SK-N-SH Human, neuroblastoma cell line

Astrocytoma Human, astrocytoma cell line

Huh-7 Human, hepatoma cell line

SW620 Human, colorectal adenocarcinoma cell line

BSMC Human, bronchial smooth muscle primary cell

HUVEC Human, umbilical vein epithelial primary cell

THP-1 Human, acute monocytic leukemia cells

C2C12, differentiated Mouse, myoblastoma line

LA-N-2 Human, neuroblastoma cell line

U-87 MG Human, glioblastomaastrocytoma cell line

C2C12, undifferentiated Mouse, myoblastoma line

MCF-7 Human, breast adenocarcinoma cell line

SK-N-AS Human, neuroblastoma cell line

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10 nM

5 nM

■ = % GAPDH Expression

siRNA-only control 40 nM

Cells only control

■ = Cell Viability

Normalized GAPDH expression and cell viability in human astrocyte primary cells. GAPDH expression and cell viability in response to decreasing concentrations of GAPDH siRNA are depicted. Cells were plated (5,000 cells per well) and transfected using the N-TER siRNA Transfection System with varying concentrations of the GAPDH siRNA (Sigma-Genosys). Twenty-four hours after transfection, cells were lysed and GAPDH mRNA levels were assessed by the QuantiGene® bDNA assay. Knockdown of GAPDH in Differentiated 3T3-L1 Cells 140

% GAPDH Expression

N-TER Peptide binds to siRNAs non-covalently at a ratio of approximately 15:1, forming an N-TER/siRNA nanoparticle that is able to rapidly cross the cell membrane and efficiently deliver its siRNA cargo into the cytoplasm.

120 100 80 60 40 20 0 30 nM

15 nM

7.5 nM

■ = % GAPDH Expression

siRNA-only control 40 nM

Cells only control

■ = Cell Viability

Normalized GAPDH expression and cell viability in differentiated 3T3-L1 adipocytes. GAPDH expression and cell viability in response to decreasing concentrations of GAPDH siRNA are depicted. Cells were plated (10,000 cells per well) and transfected using the N-TER siRNA Transfection System with varying concentrations of GAPDH siRNA (Sigma-Genosys). Twenty-four hours post-transfection, cells were lysed and GAPDH mRNA levels were assessed by the QuantiGene® bDNA assay. Selected References 1. Morris, M.C., et al., A new peptide vector for the efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res., 25, 2730-2736 (1997) 2. Simeoni, F., et al., Insight into the mechanism of the peptide-based gene delivery system MPG: Implications for delivery of siRNA into mammalian cells. Nucleic Acids Res., 31, 2717-2724 (2003) 3. Morris, K.V., et al., Small interfering RNA induced transcriptional gene silencing in human cells. Science, 305, 1289-1291 (2004) 4. Deshayes, S., et al., On mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids. Biochem. Biophys. Acta., 1667(2), 141-147 (2004) 5. Langlois, M.A., et al., Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells. J. Biol. Chem., 280(17), 16949-16954 (2005)

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RNA Isolation and Purification GenElute™ Mammalian Total RNA Miniprep Kits

TRI Reagent® RNA Isolation Reagent

The GenElute Mammalian Total RNA Miniprep Kit combines silica-membrane technology with a convenient spin column format for a rapid bind, wash, and elute method to prepare high quality total RNA.

TRI Reagent is an improved version of the single-step total RNA isolation reagent developed by Chomczynski.1 The RNA isolation method based on this reagent is widely recognized and proven for RNA applications and is supported by a substantial publication list.2 It is ideal for quick, economical, and efficient isolation of total RNA or the simultaneous isolation of RNA, DNA, and proteins from samples of human, animal, plant, yeast, bacterial and viral origin.

The resulting purified RNA is ready for Northern blots, RT-PCR and other applications for detecting knockdown at the mRNA level.

Benefits of GenElute Mammalian Total RNA Miniprep Kits: ■ ■ ■ ■ ■ ■ ■

Benefits of TRI Reagent RNA Isolation Reagent:

Purifies total RNA from up to 107 cells or 40 mg of tissue per prep Yields up to 150 μg of pure, concentrated total RNA per prep Recover RNA from as few as 100 cells Simple and efficient – 12 to 18 preps in 30 minutes Faster than gravity flow anion exchange methods No cumbersome steps associated with resins and magnetic slurries 40% more purifications than the leading supplier

■

Simultaneous isolation of RNA and protein makes knockdown detection possible at both mRNA and protein levels ■ Works with many sources: human, plant, yeast, bacterial, or viral ■ Better yields than traditional guanidine thiocyanate/cesium chloride methods Total RNA from HeLa Cells Using TRI Reagent

Ordering Information Product Description Cat. No.

Preps

Quantity

RTN10

GenElute Mammalian Total RNA Miniprep Kit

10

1 kit

RTN70

GenElute Mammalian Total RNA Miniprep Kit

70

1 kit

RTN350

GenElute Mammalian Total RNA Miniprep Kit

350

1 kit

IR ol ®

TR a

178.5

Iz

888.0

Supplier P

TR

Supplier A

e®

904.5

gm

Supplier Q

ur

904.5

IP

Sigma

Si

Average ng/ml yield from Bioanalyzer

TR

HeLa cells 7,000,000

ea

ge

nt ®

Table 1. Yield from HeLa Cells

M

Total RNA was prepared from HeLa cells using TRI Reagent from Sigma and equivalent reagents from other various suppliers. Total RNA from HeLa cells was prepared using TRIPure®, Sigma TRI Reagent®, and TRIzol®. An aliquot of total RNA was analyzed on a 1% agarose gel. RNA Marker (M) ranged from 0.2-10 kb (Cat. No. R7020). Selected References 1. Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156 (1987) 2. Chomczynski, P. and Mackey, K., Short Technical Reports. Modification of the TRI Reagent® procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechniques, 19, 924-945 (1995)

Ordering Information Cat. No. Product Description T9424

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TRI Reagent RNA, DNA and Protein Isolation Reagent

Shaping the Future of Life Science

Quantity 25 ml 100 ml 200 ml 6 × 100 ml 5

9/19/07 9:59:58 AM

Knockdown Detection–mRNA Level Quantitative Reverse Transcriptase PCR ReadyMix™ for Probe-Based Applications Probe-based qPCR relies on the sequence-specific detection of a desired PCR product. Unlike SYBR-based qPCR methods that detect all double-stranded DNA, probe-based qPCR utilizes a fluorescent-labeled target-specific probe resulting in increased specificity and sensitivity. Additionally, a variety of fluorescent dyes are available so that multiple primers can be used to simultaneously amplify many sequences. Sigma’s qRT-PCR ReadyMix combines the advantages of M-MLV Reverse Transcriptase and JumpStart Taq with a ready-to-use mix specifically designed for probe-based qRT-PCR.

SYBR Green I, a commonly used fluorescent DNA binding dye, is a cost-effective method to detect gene knockdown at the mRNA level due to the fact that it binds all double-stranded DNA and does not rely on a sequence-specific probe for detection. The SYBR Green Quantitative RT-PCR Kit delivers high reproducibility and has been optimized for use with both plate/tube real-time instruments and with the Roche LightCycler capillary instrument. A reference dye is provided in a separate vial to be used in ABI Detection Systems.

Benefits of SYBR® Green qRT-PCR Kit: ■

Benefits of qRT-PCR ReadyMix for Probe-based Applications: ■

Minimize non-specific amplification while increasing target yield and specificity, both of which result in lower, more accurate Ct values ■ Compatible with a variety of fluorescent detection methods including dual-labeled probes and Molecular Beacons, Sigma’s ReadyMix is also formulated for use on tube, plate, and capillary-based instruments Superior Sensitivity and Specificity with Sigma’s qRT-PCR ReadyMix

SYBR-based detection allows cost-effective quantitation of all double-stranded DNA ■ Optimized to help you achieve superior results, our SYBR Green qRT-PCR Kit is compatible with tube, plate, and capillarybased instruments ■ Minimize non-specific amplification while increasing target yield and specificity, both of which result in lower, more accurate Ct values Sensitive Quantitative RT-PCR Using Sigma’s SYBR Green Quantitative RT-PCR Kit

Fluorescence (F1)

10.000

10.000

Fluorescence (F1)

SYBR® Green Quantitative RT-PCR Kit

1.000

1.000

0.100

0.100

0.010

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Cycle Number 0.010

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

Cycle Number

Superior sensitivity and specificity with Sigma’s qRT-PCR ReadyMix. Quantitative RT-PCR was performed in duplicate on total RNA from the HeLa S3 cell line. Total RNA was DNase treated and diluted 10-fold in subsequent capillaries. Forward and reverse primers specific for c-Myc were used for amplification. Measurements were made using the Roche LightCycler®. Product Specifications

Product Specifications SYBR Green Taq ReadyMix for qRT-PCR M-MLV Reverse Transcriptase

Probe-based qRT-PCR ReadyMix

103 PCR Buffer

M-MLV Reverse Transcriptase

25 mM MgCl2

103 PCR Buffer

Reference Dye for Quantitative PCR

25 mM MgCl2

Ordering Information Cat. No. Product Description

Reference Dye for Quantitative PCR Ordering Information Cat. No. Product Description QR0200

6

Sensitive quantitative RT-PCR using Sigma’s SYBR Green Quantitative RT-PCR Kit. Quantitative SYBR Green RT-PCR was performed in duplicate on human total RNA from cell line HeLa S3. The total RNA was diluted 10-fold in subsequent capillaries with concentrations of 500 ng to 5 pg.

qRT-PCR ReadyMix for probe-based applications Sufficient for 100-50 μl reactions

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QR0100 Quantity

SYBR Green Quantitative RT-PCR Kit

Quantity 1 kit

1 kit

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High-Throughput Knockdown Detection–mRNA Level QuantiGene 2.0 RNA Quantification System The QuantiGene 2.0 RNA Quantification System enables highthroughput knockdown detection of specific genes and eliminates the need for RNA purification or target amplification, thereby reducing both hands-on and assay time. This platebased assay, which utilizes a unique branched DNA (bDNA) detection technology to amplify the reporter signal, provides a sensitive, high-throughput approach to quantifying transcript levels as low as 250 copies.

A549

Product Specifications Sensitivity/Limit of Quantification (LOQ): 250 copies Linear Dynamic Range: ≥ 3.5 orders of magnitude Precision: Intra- and Inter-assay CV: 10% and 15%, respectively Accuracy/Spike Recovery: 85–115%

HT29

% Expression

80 60 40 20

Em

pt

y

Co

EG nt FR ro lV F iru T ZD 6 G s( SH F B R Em C0 1 pt 01 y V Co CD ) nt K2 ro IF lV IT M ir u s ( ST 1 SH K1 Em C0 6 pt 01 y M V) Co A nt PK ro ST 3 lV YK iru 1 s( S H FO Em C0 S pt 01 y V) Co nt M JUN ro A lV PK iru 1 s ( ER 3 SH B B C0 3 01 V)

0

■

Sensitivity/Limit of Detection (LOD): 250 copies

HepG2

100

Benefits of QuantiGene 2.0: High-throughput, direct analysis of treated populations, accelerating validation of RNAi knockdown ■ Branched DNA technology amplifies the reporter signal allowing detection of low expressing genes ■ RNA levels are measured directly from crude cell lysates, reducing hands-on time involved ■ Direct measurement of mRNA from cell lysates avoids variations or errors inherent to extraction

HeLa

120

The QuantiGene system measured gene expression in comparison to empty control virus treated cells. Multiple cell lines (as noted along top x axis) were plated and grown to 80% confluency 24 hours prior to infection in 96-well plates. MISSION TRC shRNA lentiviral particles (as noted along the lower x axis) were added to the appropriate wells and the remaining wells were control samples, treated with empty vector control virus. Gene expression was measured in duplicate for each infection and results were normalized using the cyclophilin housekeeping gene. The difference in RFU signals between the infected samples and the empty control virus treated cells was used to calculate percent expression. This data show the QuantiGene system quantitates gene expression directly from crude cell lysates.

Ordering Information The QuantiGene 2.0 RNA Quantification System consists of an assay kit and probe set. For your convenience, the kits and probes can be ordered together or separately. For more information or to request a quote, please visit sigma.com/quantigene

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Knockdown Detection–Protein Level Sigma Antibodies

Examples of our application data:

Recent studies suggest that verifying knockdown at the mRNA expression level may not be enough. For true confidence in your experimental data, knockdown needs to be verified at the protein level as well. Sigma supplies the highest quality antibodies, with each meeting rigorous application testing. The performance of each of our antibodies is documented by reproducible data, including online data sheets and certificates of analysis.

Immunoblotting

MW

1

2

3

4

5

250 180 kDa 160

Benefits of Sigma Antibodies: ■

Over 4,000 antibodies available and validated to the protein of interest ■ Match antibodies to your specific application using our userfriendly online search tool ■ New antibodies added to our catalog daily ■ World-renowned distribution and technical support

Primary Antibodies ■

Cell Biology Neuroscience ■ Molecular Biology ■ Serum/Plasma Proteins ■ Recombinant Protein Purification and Detection ■

Nuclear extracts of HEK 293T cells were separated on SDS-PAGE, blotted with decreasing amounts of Rabbit Anti-DNMT1 (Cat. No. D4692), and developed with Goat anti-rabbit IgG-Peroxidase conjugate (Cat. No. A0545) using a chemiluminescent substrate. Lane 1: Antibody 2.5 μg/mL Lane 2: Antibody 1 μg/mL Lane 3: Antibody 0.5 μg/mL Lane 4: Antibody 1 μg/mL + 10 μg/mL non-relevant peptide Lane 5: Antibody 1 μg/mL + 10 μg/mL DNMT1 immunizing peptide

Cytoplasmic Staining of SNAP29

Antibody Microarrays ■

Panorama™ Antibody Microarray - XPRESS Profiler725 Kit Panorama Antibody Microarray - Cell Signaling224 Kit ■ Panorama Antibody Microarray - MAPK and PKC Pathways ■ Panorama Antibody Microarray - Gene Regulation I Kit ■ Panorama Antibody Microarray - p53 Pathways ■

Secondary Antibodies and Conjugates ■ ■ ■ ■ ■ ■

Chicken Secondary Antibodies & Conjugates Goat/Sheep Secondary Antibodies & Conjugates Human Secondary Antibodies & Conjugates Mouse/Rat Secondary Antibodies & Conjugates Rabbit Secondary Antibodies & Conjugates Other Secondary Antibodies & Conjugates

NIH3T3 cells were fixed with paraformaldheyde and stained with Anti-SNAP29 (Cat. No. S2069) at a 1:100 dilution, followed by Goat Anti-Rabbit IgG (H+L)-FITC conjugate (Cat. No. F9887).

Ordering Information

Custom Antisera Services Contact Sigma-Genosys at sigma.com/genosys and they will design and develop a customized protocol for you.

To browse our online antibody catalog or to order a printed catalog, visit our Antibody Explorer: sigma.com/antibody. 8

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Sigma siRNA Workflow Products in Action Eg5 Knockdown in HeLa Cells 120 100

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Eg5 (KIF11) is a kinesin that is involved in the formation of the mitotic spindle and in chromosome migration.3 The chemical inhibition of Eg5 activity, or siRNA mediated knockdown of Eg5 expression, leads to the formation of an abnormal spindle structure and cell cycle arrest, resulting in reduced cell proliferation.2,4 Such treatments induce easily measurable phenotypes. Cell proliferation and Eg5 expression can be quantitatively measured using commercially available tools (Figure 1). Moreover, the appearance of the cells can be qualitatively evaluated using phase contrast microscopy (Figure 2). Adherent cells exhibiting decreased Eg5 expression/activity have a rounded phenotype. This allows for the use of Eg5 as a positive control in the optimization of siRNA transfection assays.

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Figure 1. Gene expression and cell viability of HeLa cells after treatment with Eg5 siRNA

Materials and Methods Eg5 and non-targeting control siRNAs produced by Sigma. The siRNAs were transfected into HeLa cells using the N-TER™ Nanoparticle siRNA Transfection System. N-TER/siRNA complexes were transfected into HeLa cells and incubated at 37 °C, 5% CO2 overnight. After 24 hours cells were examined by phase contrast microscopy. Cells were then harvested, and gene expression was assessed using the QuantiGene® branched DNA assay (Panomics, Inc., Fremont, CA). Eg5 expression was normalized internally against that of cyclophilin B (PPIB) in each sample. Treated samples were then normalized against cell-only controls to determine Eg5 expression levels. Cell viability was assessed using a commercially available assay according to the manufacturer’s instructions.

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Results and Discussion In this experiment, a siRNA targeting the kinesin, Eg5, and a non-targeting control siRNA were transfected into HeLa cells. Treatment with the Eg5 siRNA resulted in a dose-dependent decrease in Eg5 gene expression (Figure 1). This decrease in Eg5 expression resulted in the expected rounded phenotype in the HeLa cells (Figure 2). Treatment with the non-targeting siRNA, on the other hand, had only a minor and non-specific effect on gene expression with no effect on cell viability (Figure 1). This data demonstrates the utility of the Sigma siRNA workflow product line for a typical siRNA-induced gene silencing experiment. In one stop, scientists can obtain everything that they need for their RNAi experiments. Sigma offers: ■ Custom siRNAs or pre-designed MISSION siRNAs, powered by the Rosetta siRNA Design Algorithm ■ N-TER Nanoparticle siRNA Transfection System to deliver siRNA to mammalian cells ■ QuantiGene® 2.0 RNA Quantification System for subsequent measurement of gene expression

Figure 2. Morphology of HeLa cells after treatment with 10 nM Eg5. Panels A & C: Untreated cells imaged at 40× and 100×, respectively. Panels B & D: Cells treated with Eg5 siRNA imaged at 40× and 100×, respectively. Selected References 1. Formstecher, E., et al., Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines. Oligonucleotides, 16, 387-394 (2006) 2. Mayer, T.U., et al., Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science, 286, 971-974 (1999) 3. Sawin, K.E., et al., Mitotic spindle organization by a plus-end-directed microtubule motor. Nature, 359, 540-543 (1992) 4. Weil, D., et al., Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells. Biotechniques, 33, 1244-1248 (2002)

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