mission sirna - better sirna design, better rnai performance
DESCRIPTION
A detailed product brochure on Mission siRNA - Better siRNA design, Better RNAi performance, from Sigma-Aldrich.TRANSCRIPT
■ Introduction to RNAi
■ Pre-designed siRNA for Human, Rat, and Mouse
■ Pre-arrayed siRNA Libraries
■ Custom siRNA Synthesis
■ siRNA Transfection
■ RNA Isolation and Purifi cation
■ Knockdown Detection–mRNA Level
■ High-Throughput Knockdown Detection–mRNA Level
■ Knockdown Detection–Protein Level
■ Sigma siRNA Workfl ow Products in Action
Better siRNA Design, Better RNAi Performance
X79713-siRNA brochure v2.indd cov1X79713-siRNA brochure v2.indd cov1 9/19/07 9:59:51 AM9/19/07 9:59:51 AM
ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832sigma.com/missionsirna
Introduction to RNAiRNA 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.
Processing by Dicer
RNA Interference with MISSION siRNA
Sigma siRNA Workfl ow
Order siRNADuplexes
Pre-designed MISSION® siRNAMISSION siRNA LibrariesCustom siRNA Synthesis
N-TER™ siRNATransfection System
GenElute™ Mammalian Total RNA Miniprep Kits
Quantitative RT-PCR
QuantiGene® 2.0 RNA Quantification System
Sigma Antibodies
Deliver siRNAto Cells
SelectTarget Gene
DetectKnockdown
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Our Innovation, Your Research — Shaping the Future of Life Science 1
Pre-designed siRNA for Human, Rat, and MouseMISSION 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-Specifi c Scoring Matrices (PSSM) and knowledge of the all-important siRNA seed region1 to predict the most effective and specifi c 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 real-world empirical evidence.
Silencing effi cacy 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.
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.
Benefi ts of pre-designed MISSION siRNA, powered by the Rosetta siRNA Design Algorithm:
■ Increased target specifi city, due to siRNA seed region optimization rules1
■ Effi cient knockdown of low abundance messages■ Guaranteed gene silencing■ High quality siRNA produced at ISO 9000:2001 certifi ed sites■ Freedom to operate for research use
Selected References1. 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 profi ling reveals off-target gene regulation 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 disease. Proc. Natl. Acad. Sci. USA. Nov 21;103(47):17967-72. Epub 2006 Nov 10. PMID: 17098871 [PubMed -indexed for MEDLINE] (2006)
4. Espeseth, A.S., et al., A genome wide analysis of ubiquitin ligases in APP processing identifi es a novel regulator of BACE1 mRNA levels. Mol. Cell Neurosci. Nov;33(3):227-35. Epub 2006 Sep 15. (2006)
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The MISSION siRNA Performance GuaranteeSigma guarantees that 2 out of 3 siRNA duplexes per target gene will achieve knockdown effi ciencies of greater than or equal to 75%.
Ordering InformationWe’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.
Your Favorite GeneTM
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Pre-arrayed siRNA LibrariesMISSION siRNA Libraries
MISSION siRNA Libraries offer the most compelling siRNA collec-tions for high-throughput screening by targeting genes of high therapeutic value as defi ned with input from major pharmaceu-tical companies. The fl exible format of MISSION siRNA libraries facilitates research for life scientists who are interested in specifi c classes of genes as well as those who need to generate informa-tion across the entire druggable genome.
Benefi ts of MISSION siRNA Libraries:
■ siRNA designed using the powerful Rosetta siRNA Design Algorithm, providing for more effi cient gene knockdown and greater target specifi city
■ 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 classifi cations
Product Specifi cations
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
Positive control siRNA sequences included on all plates
MISSION siRNA Human LibrariesCat. No. Product Name Targets Qty.SI00100-1SET MISSION siRNA Human 6650 1 nmol
Druggable Genome Library
SI01100-1SET MISSION siRNA Human Ligase Panel 949 1 nmol
SI02100-1SET MISSION siRNA Human Kinase Panel 714 1 nmol
SI03100-1SET MISSION siRNA Human Phosphatase 293 1 nmol Panel
SI04100-1SET MISSION siRNA Human Growth 375 1 nmol Factors and Receptors Panel
SI05100-1SET MISSION siRNA Human Cell Adhesion 496 1 nmol and Cytoskeleton Panel
SI06100-1SET MISSION siRNA Human Ion Channel 639 1 nmol and Transporters Panel
SI07100-1SET MISSION siRNA Human Assorted 228 1 nmol Function Panel
SI08100-1SET MISSION siRNA Human GPCR Panel 304 1 nmol
SI09100-1SET MISSION siRNA Human Hydrolase 204 1 nmol Panel
SI10100-1SET MISSION siRNA Human Metabolism 217 1 nmol and Cell Traffi c Panel
SI11100-1SET MISSION siRNA Human Nucleic Acid 310 1 nmol Binding Panel
SI12100-1SET MISSION siRNA Human 338 1 nmol Oxidoreductase Panel
SI13100-1SET MISSION siRNA Human Protease Panel 379 1 nmol
SI14100-1SET MISSION siRNA Human Cell Surface 722 1 nmol and Nuclear Receptors Panel
SI15100-1SET MISSION siRNA Human Cell 227 1 nmol Regulation Panel
SI16100-1SET MISSION siRNA Human Transfer and 71 1 nmol Carrier Proteins Panel
SI17100-1SET MISSION siRNA Human Transferase 184 1 nmol Panel
MISSION siRNA Rat LibrariesCat. No. Product Name Targets Qty.SI20100-1SET MISSION siRNA Rat 6025 1 nmol
Druggable Genome Library
SI21100-1SET MISSION siRNA Rat Ligase Panel 816 1 nmol
SI22100-1SET 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 342 1 nmol and Receptors Panel
SI25100-1SET MISSION siRNA Rat Cell Adhesion and 433 1 nmol Cytoskeleton Panel
SI26100-1SET MISSION siRNA Rat Ion Channel and 594 1 nmol Transporters Panel
SI27100-1SET MISSION siRNA Rat Assorted Function 210 1 nmol Panel
SI28100-1SET MISSION siRNA Rat GPCR Panel 275 1 nmol
SI29100-1SET MISSION siRNA Rat Hydrolase Panel 188 1 nmol
SI30100-1SET MISSION siRNA Rat Metabolism and 201 1 nmol Cell Traffi c Panel
SI31100-1SET MISSION siRNA Rat Nucleic 287 1 nmol Acid Binding Panel
SI32100-1SET MISSION siRNA Rat Oxidoreductase 302 1 nmol Panel
SI33100-1SET MISSION siRNA Rat Protease Panel 341 1 nmol
SI34100-1SET MISSION siRNA Rat Cell Surface and 647 1 nmol Nuclear Receptors Panel
SI35100-1SET MISSION siRNA Rat Cell Regulation 214 1 nmol Panel
SI36100-1SET MISSION siRNA Rat Transfer and Carrier 66 1 nmol Proteins Panel
SI37100-1SET MISSION siRNA Rat Transferase Panel 174 1 nmol
MISSION siRNA Mouse LibrariesCat. No. Product Name Targets Qty.SI42050-1SET MISSION siRNA Mouse Kinase Panel 623 500 pmol
Ordering Information
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Our Innovation, Your Research — Shaping the Future of Life Science 3
Custom siRNA SynthesissiRNA Synthesis According to Your Specifi cation
Don’t see a pre-designed MISSION siRNA that will work for you? Scientists at Sigma have identifi ed 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 effi ciency and faster deprotection, resulting in higher quality siRNA synthesis and faster turn-around times.
Benefi ts 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 purifi cations■ High-throughput capacity for large projects
Guaranteed Yields and Transfections 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
Transfections (approx.)
24-well plate format
150 wells
375 wells
750 wells
3750 wells
75 wells
375 wells
750 wells
3750 wells
Please inquire for alternative quantities, purifi cation grades and labels.
Turn-Around Time for siRNA OligosDesalted 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
Turn-around time (TAT)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
Sigma’s siRNA is produced at sites around the world, providing for fi rst-class turn-around times and customer service.
Ordering InformationThose wishing to have siRNA synthesized according to their own design and specifi cations may do so at:sigma.com/custom_sirna.
Simply use the Sigma siRNA confi guration tools to supply us with:■ Your sequence to be synthesized■ Amount to be synthesized■ siRNA Purifi cation Level desired: • Standard Desalted • HPLC purifi cation • PAGE purifi cation ■ Number of tubes to aliquot into■ Modifi cations desired: • 5’ and 3’ Labels – 6-FAM – Amine – Biotin – Cy®3 – Cy5 – Cy5.5 – Fluorescein – Phosphate • Internal Modifi cations – 2’ O-Methyl RNA – LNA
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4 ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832sigma.com/missionsirna
N-TER™ Nanoparticle siRNA Transfection System
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 non-dividing cells.
Sigma’s N-TER Nanoparticle siRNA Transfection System is based on a peptide transfection reagent specifi cally designed to bypass these limitations and allow for effi cient delivery of siRNAs into these historically recalcitrant eukaryotic cell types.
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 effi ciently deliver its siRNA cargo into the cytoplasm.
Benefi ts of the N-TER Nanoparticle siRNA Transfection System:
■ Effi cient 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 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:3T3-L1, differentiated Mouse, embryonic fi broblast cell line
HEK293THuman, embryonic kidney cell line
MDA-MB-231Human, breast adeno carcinoma cell line
A2780 Human, ovarian carcinoma cell line
HeLaHuman, cervical adeno carcinoma cell line
NHAHuman, astrocyte primary cell
A431Human, ovarian carcinoma cell line
HepatocyteRat, hepatocyte primary cell
NHEK-ADHuman, adult keratinacyte primary cell
A549Human, lung carcinoma cell line
HepG2Human, hepatocarcinoma cell line
RAW264.7Mouse, macrophage cell line
ASPC-1Human, pancreatic carcinoma cell line
HT-29Human, colorectal adenocarcinoma cell line
SK-N-SHHuman, neuroblastoma cell line
AstrocytomaHuman, astrocytoma cell line
Huh-7Human, hepatoma cell line
SW620Human, colorectal adenocarcinoma cell line
BSMCHuman, bronchial smooth muscle primary cell
HUVECHuman, umbilical vein epithelial primary cell
THP-1Human, acute monocytic leukemia cells
C2C12, differentiated Mouse, myoblastoma line
LA-N-2Human, neuroblastoma cell line
U-87 MGHuman, glioblastoma-astrocytoma cell line
C2C12, undifferentiated Mouse, myoblastoma line
MCF-7Human, breast adeno carcinoma cell line
SK-N-ASHuman, neuroblastoma cell line
siRNA Transfection
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.
Cells onlycontrol
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■ = % GAPDH Expression ■ = Cell Viability
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Normalized GAPDH expression and cell viability in differ-entiated 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 trans-fected 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 References1. Morris, M.C., et al., A new peptide vector for the effi cient 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)
Knockdown of GAPDH in Differentiated 3T3-L1 Cells
Cells onlycontrol
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■ = % GAPDH Expression ■ = Cell Viability
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Our Innovation, Your Research — Shaping the Future of Life Science 5
RNA Isolation and Purifi cation 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 purifi ed RNA is ready for Northern blots, RT-PCR and other applications for detecting knockdown at the mRNA level.
Benefi ts of GenElute Mammalian Total RNA Miniprep Kits:
■ Purifi es 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 effi cient – 12 to 18 preps in 30 minutes■ Faster than gravity fl ow anion exchange methods■ No cumbersome steps associated with resins and magnetic slurries■ 40% more purifi cations than the leading supplier
Table 1. Yield from HeLa Cells
HeLa cells Average ng/ml yield 7,000,000 from Bioanalyzer
Sigma 904.5
Supplier Q 904.5
Supplier A 888.0
Supplier P 178.5
Ordering InformationCat. 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
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 effi cient isolation of total RNA or the simultaneous isolation of RNA, DNA, and proteins from samples of human, animal, plant, yeast, bacterial and viral origin.
Benefi ts 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
TRIPu
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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 References1. 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. Modifi cation of the TRI Reagent® procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. BioTechniques, 19, 924-945 (1995)
Ordering InformationCat. No. Product Description Quantity
T9424 TRI Reagent RNA, DNA and 25 ml Protein Isolation Reagent 100 ml 200 ml 6 × 100 ml
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6 ORDER: 800-325-3010 TECHNICAL SERVICE: 800-325-5832sigma.com/missionsirna
Knockdown Detection–mRNA LevelQuantitative Reverse Transcriptase PCR ReadyMix™ for Probe-Based Applications
Probe-based qPCR relies on the sequence-specifi c detection of a desired PCR product. Unlike SYBR-based qPCR methods that detect all double-stranded DNA, probe-based qPCR utilizes a fl uorescent-labeled target-specifi c probe resulting in increased specifi city and sensitivity. Additionally, a variety of fl uorescent dyes are available so that multiple primers can be used to simul-taneously 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 specifi cally designed for probe-based qRT-PCR.
Benefi ts of qRT-PCR ReadyMix for Probe-based Applications:■ Minimize non-specifi c amplifi cation while increasing target
yield and specifi city, both of which result in lower, more accurate Ct values
■ Compatible with a variety of fl uorescent 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 Specifi city with Sigma’s qRT-PCR ReadyMix
Cycle Number0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
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Superior sensitivity and specifi city 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 specifi c for c-Myc were used for amplifi ca-tion. Measurements were made using the Roche LightCycler®.
Product Specifi cations
Probe-based qRT-PCR ReadyMix
M-MLV Reverse Transcriptase
103 PCR Buffer
25 mM MgCl2
Reference Dye for Quantitative PCR
Ordering InformationCat. No. Product Description Quantity
QR0200 qRT-PCR ReadyMix for 1 kit probe-based applications Suffi cient for 100-50 μl reactions
SYBR® Green Quantitative RT-PCR Kit
SYBR Green I, a commonly used fl uorescent 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-specifi c probe for detection.
The SYBR Green Quantitative RT-PCR Kit delivers high reprodu-cibility 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.
Benefi ts of SYBR® Green qRT-PCR Kit:■ 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 capillary-based instruments
■ Minimize non-specifi c amplifi cation while increasing target yield and specifi city, both of which result in lower, more accurate Ct values
Sensitive Quantitative RT-PCR Using Sigma’s SYBR Green Quantitative RT-PCR Kit
Cycle Number12 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
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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.
Product Specifi cations
SYBR Green Taq ReadyMix for qRT-PCR
M-MLV Reverse Transcriptase
103 PCR Buffer
25 mM MgCl2
Reference Dye for Quantitative PCR
Ordering InformationCat. No. Product Description Quantity
QR0100 SYBR Green Quantitative 1 kit RT-PCR Kit
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Our Innovation, Your Research — Shaping the Future of Life Science 7
QuantiGene 2.0 RNA Quantifi cation System
The QuantiGene 2.0 RNA Quantifi cation System enables high-throughput knockdown detection of specifi c genes and elimi-nates the need for RNA purifi cation or target amplifi cation, thereby reducing both hands-on and assay time. This plate-based 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.
Benefi ts of QuantiGene 2.0:
■ High-throughput, direct analysis of treated populations, accelerating validation of RNAi knockdown
■ Branched DNA technology amplifi es 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
Product Specifi cations
Sensitivity/Limit of Detection (LOD): 250 copies
Sensitivity/Limit of Quantifi cation (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%
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% confl uency 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 InformationThe QuantiGene 2.0 RNA Quantifi cation 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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High-Throughput Knockdown Detection–mRNA Level
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Sigma Antibodies
Recent studies suggest that verifying knockdown at the mRNA expression level may not be enough. For true confi dence in your experimental data, knockdown needs to be verifi ed 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 certifi cates of analysis.
Benefi ts of Sigma Antibodies:
■ Over 4,000 antibodies available and validated to the protein of interest
■ Match antibodies to your specifi c application using our user-friendly online search tool
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Primary Antibodies
■ Cell Biology■ Neuroscience■ Molecular Biology■ Serum/Plasma Proteins■ Recombinant Protein Purifi cation and Detection
Antibody Microarrays
■ Panorama™ Antibody Microarray - XPRESS Profi ler725 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
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Contact Sigma-Genosys at sigma.com/genosys and they will design and develop a customized protocol for you.
Examples of our application data:
Immunoblotting
250
160
1 2 3MW
180 kDa
4 5
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/mLLane 2: Antibody 1 μg/mLLane 3: Antibody 0.5 μg/mLLane 4: Antibody 1 μg/mL + 10 μg/mL non-relevant peptideLane 5: Antibody 1 μg/mL + 10 μg/mL DNMT1 immunizing
peptide
Cytoplasmic Staining of SNAP29
NIH3T3 cells were fi xed 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
To browse our online antibody catalog or to order a printed catalog, visit our Antibody Explorer: sigma.com/antibody.
Knockdown Detection–Protein Level
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Our Innovation, Your Research — Shaping the Future of Life Science 9
Sigma siRNA Workfl ow Products in ActionFunctional Characterization of Eg5 in HeLa Cells
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 struc-ture 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.
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.
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-specifi c effect on gene expression with no effect on cell viability (Figure 1).
This data demonstrates the utility of the Sigma siRNA workfl ow 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 Quantifi cation 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.
A B
C D
Figure 1. Gene expression and cell viability of HeLa cells after treatment with Eg5 siRNA
Selected References1. Formstecher, E., et al., Combination of active and inactive siRNA targeting
the mitotic kinesin Eg5 impairs silencing effi ciency in several cancer cell lines. Oligonucleotides, 16, 387-394 (2006)
2. Mayer, T.U., et al., Small molecule inhibitor of mitotic spindle bipolarity identifi ed 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)
Eg5 Knockdown in HeLa Cells
5K cell/well 10K cell/well 15K cell/well
[siRNA] (nM)
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iability
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Eg5 Neg. Ctrl.
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