Better siRNA Design,

Better RNAi Performance

■ Introduction to RNAi
■ Pre-designed siRNA for Human,
Rat, and Mouse
■ Pre-arrayed siRNA Libraries
■ Custom siRNA Synthesis
■ siRNA Transfection
■ RNA Isolation and Purification
■ Knockdown Detection–
mRNA Level
■ High-Throughput Knockdown
Detection–mRNA Level
■ Knockdown Detection–
Protein Level
■ Sigma siRNA Workflow Products
in Action

X79713-siRNA brochure v2.indd cov1 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 Order siRNA Deliver siRNA Detect

Target Gene Duplexes to Cells Knockdown

Pre-designed MISSION® siRNA N-TER™ siRNA GenElute™ Mammalian Total

MISSION siRNA Libraries
Custom siRNA Synthesis
Transfection System
RNA Miniprep Kits
Quantitative RT-PCR
QuantiGene® 2.0 RNA
Quantification System
Sigma Antibodies

sigma.com/missionsirna ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

X79713-siRNA brochure v2.indd cov2 9/19/07 9:59:54 AM

 Pre-designed siRNA for Human, Rat, and Mouse
MISSION siRNA Using a siRNA designed with a best-in-class algorithm saves
time and money, enabling you to focus on downstream
Current studies suggest that the nucleotide sequence of an
applications, not up-front siRNA design and validation work.
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 Benefits of pre-designed MISSION siRNA,
partnership with Rosetta Inpharmatics, a leader in advanced powered by the Rosetta siRNA Design Algorithm:
siRNA research, to use the proprietary Rosetta siRNA Design
■ Increased target specificity, due to siRNA seed region
Algorithm to design its MISSION line of siRNA. The Rosetta
optimization rules1
siRNA Design Algorithm utilizes Position-Specific Scoring
■ Efficient knockdown of low abundance messages
Matrices (PSSM) and knowledge of the all-important siRNA
■ Guaranteed gene silencing
seed region1 to predict the most effective and specific siRNA
■ High quality siRNA produced at ISO 9000:2001 certified sites
sequences for your target gene of interest. Additionally, the
■ Freedom to operate for research use
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 real- The MISSION siRNA Performance Guarantee
Sigma guarantees that 2 out of 3 siRNA duplexes
world empirical evidence.
per target gene will achieve knockdown efficiencies
of greater than or equal to 75%.

100
90
% Expression Remaining

80
70
60
50
40
30
20
10
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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 Ordering Information

1. Jackson, A.L., et al., Widespread siRNA “off-target” transcript silencing
mediated by seed region sequence complementarity. RNA, 12, 1179-1187 We’ve made ordering MISSION siRNA for single gene targets
(2006)
easy through Sigma’s state-of-the-art Web interface, Your
2. Jackson, A.L., et al., Expression profiling reveals off-target gene regulation
Favorite Gene at sigma.com/yfg.
by RNAi. Nat. Biotechnol., 21, 635-637 (2003)
3. Majercak, J., et al., LRRTM3 promotes processing of amyloid-precursor pro-
tein by BACE1 and is a positional candidate gene for late-onset Alzheimer’s Simply search against your gene of interest by gene name,
disease. Proc. Natl. Acad. Sci. USA. Nov 21;103(47):17967-72. Epub 2006 symbol, RefSeq, or Gene ID number. For each gene search you
Nov 10. PMID: 17098871 [PubMed -indexed for MEDLINE] (2006) 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 Your Favorite Gene TM

Neurosci. Nov;33(3):227-35. Epub 2006 Sep 15. (2006)

Our Innovation, Your Research — Shaping the Future of Life Science 1

X79713-siRNA brochure v2.indd 1 9/19/07 9:59:54 AM

 Pre-arrayed siRNA Libraries
MISSION siRNA Libraries
MISSION siRNA Libraries offer the most compelling siRNA collec-
tions for high-throughput screening by targeting genes of high
therapeutic value as defined with input from major pharmaceu-
tical 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 informa-
tion across the entire druggable genome.
Benefits of MISSION siRNA Libraries:
■ 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 pharmaceuti-
cal 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 MISSION siRNA Rat Libraries

Cat. No. Product Name Targets Qty. Cat. No. Product Name Targets Qty.
SI00100-1SET MISSION siRNA Human 6650 1 nmol SI20100-1SET MISSION siRNA Rat 6025 1 nmol
Druggable Genome Library Druggable Genome Library
SI01100-1SET MISSION siRNA Human Ligase Panel 949 1 nmol SI21100-1SET MISSION siRNA Rat Ligase Panel 816 1 nmol
SI02100-1SET MISSION siRNA Human Kinase Panel 714 1 nmol SI22100-1SET MISSION siRNA Rat Kinase Panel 669 1 nmol
SI03100-1SET MISSION siRNA Human Phosphatase 293 1 nmol SI23100-1SET MISSION siRNA Rat Phosphatase Panel 266 1 nmol
Panel SI24100-1SET MISSION siRNA Rat Growth Factors 342 1 nmol
SI04100-1SET MISSION siRNA Human Growth 375 1 nmol and Receptors Panel
Factors and Receptors Panel SI25100-1SET MISSION siRNA Rat Cell Adhesion and 433 1 nmol
SI05100-1SET MISSION siRNA Human Cell Adhesion 496 1 nmol Cytoskeleton Panel
and Cytoskeleton Panel SI26100-1SET MISSION siRNA Rat Ion Channel and 594 1 nmol
SI06100-1SET MISSION siRNA Human Ion Channel 639 1 nmol Transporters Panel
and Transporters Panel SI27100-1SET MISSION siRNA Rat Assorted Function 210 1 nmol
SI07100-1SET MISSION siRNA Human Assorted 228 1 nmol Panel
Function Panel SI28100-1SET MISSION siRNA Rat GPCR Panel 275 1 nmol
SI08100-1SET MISSION siRNA Human GPCR Panel 304 1 nmol SI29100-1SET MISSION siRNA Rat Hydrolase Panel 188 1 nmol
SI09100-1SET MISSION siRNA Human Hydrolase 204 1 nmol SI30100-1SET MISSION siRNA Rat Metabolism and 201 1 nmol
Panel Cell Traffic Panel
SI10100-1SET MISSION siRNA Human Metabolism 217 1 nmol SI31100-1SET MISSION siRNA Rat Nucleic 287 1 nmol
and Cell Traffic Panel Acid Binding Panel
SI11100-1SET MISSION siRNA Human Nucleic Acid 310 1 nmol SI32100-1SET MISSION siRNA Rat Oxidoreductase 302 1 nmol
Binding Panel Panel
SI12100-1SET MISSION siRNA Human 338 1 nmol SI33100-1SET MISSION siRNA Rat Protease Panel 341 1 nmol
Oxidoreductase Panel
SI34100-1SET MISSION siRNA Rat Cell Surface and 647 1 nmol
SI13100-1SET MISSION siRNA Human Protease Panel 379 1 nmol Nuclear Receptors Panel
SI14100-1SET MISSION siRNA Human Cell Surface 722 1 nmol SI35100-1SET MISSION siRNA Rat Cell Regulation 214 1 nmol
and Nuclear Receptors Panel Panel
SI15100-1SET MISSION siRNA Human Cell 227 1 nmol SI36100-1SET MISSION siRNA Rat Transfer and Carrier 66 1 nmol
Regulation Panel Proteins Panel
SI16100-1SET MISSION siRNA Human Transfer and 71 1 nmol SI37100-1SET MISSION siRNA Rat Transferase Panel 174 1 nmol
Carrier Proteins Panel
SI17100-1SET MISSION siRNA Human Transferase 184 1 nmol MISSION siRNA Mouse Libraries
Panel Cat. No. Product Name Targets Qty.
SI42050-1SET MISSION siRNA Mouse Kinase Panel 623 500 pmol

2 sigma.com/missionsirna ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

X79713-siRNA brochure v2.indd 2 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 ribo-

nucleoside 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,
Sigma’s siRNA is produced at sites around the world, providing
resulting in higher quality siRNA synthesis and faster turn-
for first-class turn-around times and customer service.
around times.

Benefits of Sigma’s Custom siRNA Synthesis Service:

Ordering Information
■ High-quality and cost-effective siRNA synthesis
■ Fast turn-around times Those wishing to have siRNA synthesized according to
■ Reduced need for costly and time-consuming purifications their own design and specifications may do so at:
■ High-throughput capacity for large projects sigma.com/custom_sirna.

Guaranteed Yields and Transfections for Simply use the Sigma siRNA configuration tools to supply us with:
■ Your sequence to be synthesized
siRNA Oligos
■ Amount to be synthesized
Desalted PAGE RP-HPLC ■ siRNA Purification Level desired:
Yields • Standard Desalted
Guaranteed • HPLC purification
2 5 10 50 1 5 10 50
yield (OD)
• PAGE purification
Approx. yield ■ Number of tubes to aliquot into
10 25 50 250 5 25 50 250
(nmols*)
■ Modifications desired:
Transfections (approx.)
• 5’ and 3’ Labels
24-well plate 150 375 750 3750 75 375 750 3750
format wells wells wells wells wells wells wells wells – 6-FAM
Please inquire for alternative quantities, purification grades and labels.
– Amine
– Biotin
Turn-Around Time for siRNA Oligos – Cy®3
– Cy5
Desalted PAGE RP-HPLC
– Cy5.5
Yields
– Fluorescein
Guaranteed
2 5 10 50 1 5 10 50 – Phosphate
yield (OD)
• Internal Modifications
Approx. yield
10 25 50 250 5 25 50 250 – 2’ O-Methyl RNA
(nmols*)
Turn-around time (TAT) – LNA
Single-strand, unlabeled
No. of days 4 4 5 5 6 8 8 8
Single-strand, labeled
No. of days 5 5 6 6 7 9 9 9
Guaranteed duplex, unlabeled
No. of days 5 5 6 6 7 9 9 9
Guaranteed duplex, labeled
No. of days 6 6 7 7 8 10 10 10
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 — Shaping the Future of Life Science 3

X79713-siRNA brochure v2.indd 3 9/19/07 9:59:57 AM

 siRNA Transfection
N-TER™ Nanoparticle siRNA Transfection System Knockdown of GAPDH in Human Astrocyte
Primary Cells
Traditional lipid-based siRNA transfection reagents exhibit a number 100
of drawbacks, including a limited ability to transfect into a variety

% GAPDH Expression
of cell types, such as primary, neuronal, differentiated, and non- 80

dividing cells.
60

Sigma’s N-TER Nanoparticle siRNA Transfection System is based 40

on a peptide transfection reagent specifically designed to bypass
20
these limitations and allow for efficient delivery of siRNAs into
these historically recalcitrant eukaryotic cell types. 0
20 nM 10 nM 5 nM siRNA-only Cells only
control control
N-TER Peptide binds to siRNAs non-covalently at a ratio of 40 nM

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.
Benefits of the N-TER Nanoparticle siRNA
Transfection System:
■ Efficient transfection of a wide variety of historically hard-to-
transfect 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
■ = % GAPDH Expression ■ = 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 concen-
trations 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

experiments targeting the same gene

120
■ Fast and simple protocol easily adapted for high throughput
100
and reverse transfection applications
80
For more information, visit us online at sigma.com/nter. 60
40
N-TER has been validated to work in these cell types: 20
3T3-L1, HEK293T MDA-MB-231 0
differentiated Mouse, Human, embryonic kidney Human, breast 30 nM 15 nM 7.5 nM siRNA-only Cells only
embryonic fibroblast cell line cell line adenocarcinoma cell line control control
40 nM
A2780 HeLa NHA
■ = % GAPDH Expression ■ = Cell Viability
Human, ovarian carcinoma Human, cervical Human, astrocyte
cell line adenocarcinoma cell line primary cell
Normalized GAPDH expression and cell viability in differ-
A431 Hepatocyte NHEK-AD entiated 3T3-L1 adipocytes. GAPDH expression and cell viability
Human, ovarian carcinoma Rat, hepatocyte primary cell Human, adult keratinacyte in response to decreasing concentrations of GAPDH siRNA are
cell line primary cell
depicted. Cells were plated (10,000 cells per well) and trans-
A549 HepG2 RAW264.7 fected using the N-TER siRNA Transfection System with varying
Human, lung carcinoma Human, hepatocarcinoma Mouse, macrophage concentrations of GAPDH siRNA (Sigma-Genosys). Twenty-four
cell line cell line cell line hours post-transfection, cells were lysed and GAPDH mRNA
ASPC-1 HT-29 SK-N-SH levels were assessed by the QuantiGene® bDNA assay.
Human, pancreatic Human, colorectal Human, neuroblastoma
carcinoma cell line adenocarcinoma cell line cell line Selected References
1. Morris, M.C., et al., A new peptide vector for the efficient delivery of
Astrocytoma Huh-7 SW620
oligonucleotides into mammalian cells. Nucleic Acids Res., 25, 2730-2736
Human, astrocytoma Human, hepatoma cell line Human, colorectal
(1997)
cell line adenocarcinoma cell line
2. Simeoni, F., et al., Insight into the mechanism of the peptide-based gene
BSMC HUVEC THP-1 delivery system MPG: Implications for delivery of siRNA into mammalian
Human, bronchial smooth Human, umbilical vein Human, acute monocytic cells. Nucleic Acids Res., 31, 2717-2724 (2003)
muscle primary cell epithelial primary cell leukemia cells 3. Morris, K.V., et al., Small interfering RNA induced transcriptional gene
silencing in human cells. Science, 305, 1289-1291 (2004)
C2C12, LA-N-2 U-87 MG
4. Deshayes, S., et al., On mechanism of non-endosomial peptide-mediated
differentiated Mouse, Human, neuroblastoma Human, glioblastoma-
cellular delivery of nucleic acids. Biochem. Biophys. Acta., 1667(2),
myoblastoma line cell line astrocytoma cell line
141-147 (2004)
C2C12, MCF-7 SK-N-AS 5. Langlois, M.A., et al., Cytoplasmic and nuclear retained DMPK mRNAs are
undifferentiated Mouse, Human, breast Human, neuroblastoma targets for RNA interference in myotonic dystrophy cells. J. Biol. Chem.,
myoblastoma line adenocarcinoma cell line cell line 280(17), 16949-16954 (2005)

4 sigma.com/missionsirna ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

X79713-siRNA brochure v2.indd 4 9/19/07 9:59:57 AM

 RNA Isolation and Purification
GenElute™ Mammalian Total RNA Miniprep Kits
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.
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:
■ 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
Table 1. Yield from HeLa Cells
TRI Reagent® RNA Isolation Reagent
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 publica-
tion 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.
Benefits of TRI Reagent RNA Isolation Reagent:
■ 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
nt ®
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ea

HeLa cells Average ng/ml yield

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Supplier Q 904.5
Supplier A 888.0
Supplier P 178.5

Ordering Information
Cat. No. Product Description Preps Quantity
RTN10 GenElute Mammalian 10 1 kit
Total RNA Miniprep Kit
RTN70 GenElute Mammalian 70 1 kit
Total RNA Miniprep Kit
RTN350 GenElute Mammalian 350 1 kit
Total RNA Miniprep Kit 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 Quantity
T9424 TRI Reagent RNA, DNA and 25 ml
Protein Isolation Reagent 100 ml
200 ml
6 × 100 ml

Our Innovation, Your Research — Shaping the Future of Life Science 5

X79713-siRNA brochure v2.indd 5 9/19/07 9:59:58 AM

 Knockdown Detection–mRNA Level
Quantitative Reverse Transcriptase PCR ReadyMix™ SYBR® Green Quantitative RT-PCR Kit
for Probe-Based Applications SYBR Green I, a commonly used fluorescent DNA binding dye, is
Probe-based qPCR relies on the sequence-specific detection of a cost-effective method to detect gene knockdown at the mRNA
a desired PCR product. Unlike SYBR-based qPCR methods that level due to the fact that it binds all double-stranded DNA and
detect all double-stranded DNA, probe-based qPCR utilizes a does not rely on a sequence-specific probe for detection.
fluorescent-labeled target-specific probe resulting in increased
specificity and sensitivity. Additionally, a variety of fluorescent The SYBR Green Quantitative RT-PCR Kit delivers high reprodu-
dyes are available so that multiple primers can be used to simul- cibility and has been optimized for use with both plate/tube
taneously amplify many sequences. real-time instruments and with the Roche LightCycler capillary
instrument. A reference dye is provided in a separate vial to be
Sigma’s qRT-PCR ReadyMix combines the advantages of M-MLV used in ABI Detection Systems.
Reverse Transcriptase and JumpStart Taq with a ready-to-use mix
specifically designed for probe-based qRT-PCR. Benefits of SYBR® Green qRT-PCR Kit:
■ SYBR-based detection allows cost-effective quantitation of all
Benefits of qRT-PCR ReadyMix for double-stranded DNA
Probe-based Applications: ■ Optimized to help you achieve superior results, our SYBR Green
qRT-PCR Kit is compatible with tube, plate, and capillary-
■ Minimize non-specific amplification while increasing target
based instruments
yield and specificity, both of which result in lower, more
■ Minimize non-specific amplification while increasing target
accurate Ct values
yield and specificity, both of which result in lower, more
■ Compatible with a variety of fluorescent detection methods
accurate Ct values
including dual-labeled probes and Molecular Beacons, Sigma’s
ReadyMix is also formulated for use on tube, plate, and
capillary-based instruments Sensitive Quantitative RT-PCR Using Sigma’s
SYBR Green Quantitative RT-PCR Kit
Superior Sensitivity and Specificity with 10.000

Sigma’s qRT-PCR ReadyMix

Fluorescence (F1)

10.000
1.000
Fluorescence (F1)

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 Sensitive quantitative RT-PCR using Sigma’s SYBR Green

Quantitative RT-PCR Kit. Quantitative SYBR Green RT-PCR was
Superior sensitivity and specificity with Sigma’s qRT-PCR performed in duplicate on human total RNA from cell line HeLa S3.
ReadyMix. Quantitative RT-PCR was performed in duplicate The total RNA was diluted 10-fold in subsequent capillaries with
on total RNA from the HeLa S3 cell line. Total RNA was DNase concentrations of 500 ng to 5 pg.
treated and diluted 10-fold in subsequent capillaries. Forward
and reverse primers specific for c-Myc were used for amplifica- Product Specifications
tion. Measurements were made using the Roche LightCycler®.
SYBR Green Taq ReadyMix for qRT-PCR
Product Specifications 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
Reference Dye for Quantitative PCR
Cat. No. Product Description Quantity
Ordering Information QR0100 SYBR Green Quantitative 1 kit
RT-PCR Kit
Cat. No. Product Description Quantity
QR0200 qRT-PCR ReadyMix for 1 kit
probe-based applications
Sufficient for 100-50 μl reactions

6 sigma.com/missionsirna ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

X79713-siRNA brochure v2.indd 6 9/19/07 9:59:58 AM

 High-Throughput Knockdown Detection–mRNA Level
QuantiGene 2.0 RNA Quantification System
The QuantiGene 2.0 RNA Quantification System enables high- A549 HeLa HepG2 HT29
120
throughput knockdown detection of specific genes and elimi-
100
nates the need for RNA purification or target amplification,

% Expression
thereby reducing both hands-on and assay time. This plate- 80

based assay, which utilizes a unique branched DNA (bDNA) 60

detection technology to amplify the reporter signal, provides a 40
sensitive, high-throughput approach to quantifying transcript
20
levels as low as 250 copies.
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■ 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
Product Specifications
Sensitivity/Limit of Detection (LOD): 250 copies
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%
Em
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 calcu-
late 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

Our Innovation, Your Research — Shaping the Future of Life Science 7

X79713-siRNA brochure v2.indd 7 9/19/07 9:59:58 AM

 Knockdown Detection–Protein Level
Sigma Antibodies Examples of our application data:
Recent studies suggest that verifying knockdown at the mRNA Immunoblotting
expression level may not be enough. For true confidence in your
experimental data, knockdown needs to be verified at the protein MW 1 2 3 4 5
level as well. Sigma supplies the highest quality antibodies, with
each meeting rigorous application testing. The performance 250
of each of our antibodies is documented by reproducible data, 180 kDa
including online data sheets and certificates of analysis.

Benefits of Sigma Antibodies: 160

■ Over 4,000 antibodies available and validated to the protein
of interest
■ Match antibodies to your specific application using our user- Nuclear extracts of HEK 293T cells were separated on
SDS-PAGE, blotted with decreasing amounts of Rabbit
friendly online search tool
Anti-DNMT1 (Cat. No. D4692), and developed with Goat
■ New antibodies added to our catalog daily
anti-rabbit IgG-Peroxidase conjugate (Cat. No. A0545)
■ World-renowned distribution and technical support
using a chemiluminescent substrate.
Lane 1: Antibody 2.5 μg/mL
Primary Antibodies Lane 2: Antibody 1 μg/mL
■ Cell Biology Lane 3: Antibody 0.5 μg/mL
■ Neuroscience Lane 4: Antibody 1 μg/mL + 10 μg/mL non-relevant peptide
■ Molecular Biology Lane 5: Antibody 1 μg/mL + 10 μg/mL DNMT1 immunizing
peptide
■ Serum/Plasma Proteins
■ Recombinant Protein Purification and Detection 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 sigma.com/missionsirna ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832

X79713-siRNA brochure v2.indd 8 9/19/07 9:59:59 AM

 Sigma siRNA Workflow Products in Action
Functional Characterization of Eg5 in HeLa Cells Eg5 Knockdown in HeLa Cells
Eg5 (KIF11) is a kinesin that is involved in the formation of the 120
mitotic spindle and in chromosome migration.3 The chemical

Percent Gen e Expression

100 100

Percent Cell Viability

inhibition of Eg5 activity, or siRNA mediated knockdown of Eg5
expression, leads to the formation of an abnormal spindle struc- 80 80

ture and cell cycle arrest, resulting in reduced cell proliferation.2,4 60 60

Such treatments induce easily measurable phenotypes. Cell 40 40

proliferation and Eg5 expression can be quantitatively measured
20 20
using commercially available tools (Figure 1). Moreover, the
appearance of the cells can be qualitatively evaluated using 0 0
20 10 20 Cells–Only
phase contrast microscopy (Figure 2). Adherent cells exhibiting Eg5 Neg. Ctrl.
decreased Eg5 expression/activity have a rounded phenotype. [siRNA] (nM)
This allows for the use of Eg5 as a positive control in the
optimization of siRNA transfection assays. 5K cell/well 10K cell/well 15K cell/well

Figure 1. Gene expression and cell viability of HeLa cells

Materials and Methods after treatment with Eg5 siRNA
Eg5 and non-targeting control siRNAs produced by Sigma.
The siRNAs were transfected into HeLa cells using the N-TER™ A B
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
C D
available assay according to the manufacturer’s instructions.

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, Figure 2. Morphology of HeLa cells after treatment with
on the other hand, had only a minor and non-specific effect on 10 nM Eg5. Panels A & C: Untreated cells imaged at 40× and
gene expression with no effect on cell viability (Figure 1). 100×, respectively. Panels B & D: Cells treated with Eg5 siRNA
imaged at 40× and 100×, respectively.
This data demonstrates the utility of the Sigma siRNA workflow
product line for a typical siRNA-induced gene silencing Selected References
experiment. In one stop, scientists can obtain everything that 1. Formstecher, E., et al., Combination of active and inactive siRNA targeting
they need for their RNAi experiments. Sigma offers: the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell
lines. Oligonucleotides, 16, 387-394 (2006)
■ Custom siRNAs or pre-designed MISSION siRNAs, powered by
the Rosetta siRNA Design Algorithm 2. Mayer, T.U., et al., Small molecule inhibitor of mitotic spindle bipolarity
identified in a phenotype-based screen. Science, 286, 971-974 (1999)
■ N-TER Nanoparticle siRNA Transfection System to deliver
siRNA to mammalian cells 3. Sawin, K.E., et al., Mitotic spindle organization by a plus-end-directed
microtubule motor. Nature, 359, 540-543 (1992)
■ QuantiGene® 2.0 RNA Quantification System for subsequent
4. Weil, D., et al., Targeting the kinesin Eg5 to monitor siRNA transfection in
measurement of gene expression
mammalian cells. Biotechniques, 33, 1244-1248 (2002)

Our Innovation, Your Research — Shaping the Future of Life Science 9

X79713-siRNA brochure v2.indd 9 9/19/07 9:59:59 AM

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