SureSilencing sirna Array Technology Overview Pathway-Focused sirna-based RNA Interference
Topics to be Covered Who is SuperArray? Brief Introduction to RNA Interference Challenges Facing RNA Interference Researchers Solutions SureSilencing sirna Arrays Plasmids Provide How SureSilencing sirna Arrays Works 96-Well Optical Cell-Culture Plate 42 Genes plus Replicate RNAi and Assay Controls Simple Protocol Validated Knockdown How YOU Can Use SureSilencing sirna Arrays Apoptosis and NFκB Signaling Application Examples
Who IS SuperArray? Located in Frederick, Maryland, USA ~ 30 miles north of the NIH Bethesda campus Founded 9 years ago by scientists working at the NIH > 30 % of employees are PhDs in many departments Growth through new product development and product line extensions Core Values: Innovation, Focus, Systematic Design Goal: Transform cutting-edge biological information and technologies into easy to use, accessible, and innovative system biology research tools.
4 Stages of Gene Expression Research 1 Discovery High-Density, Genome-Wide 2 Screening Low-Density, Pathway-Focused 3 Confirmation Real-Time RT-PCR Validation 4 Gene Function Protein Function 1 Low sample number, High gene content Agilent Microarray Services 2 Higher sample number, Lower gene number RT 2 Profiler PCR Array and Oligo GEArray DNA Microarrays 3 Very low gene number, Very high value genes RT 2 qpcr Assays & Master Mixes 4 RNA Interference & Functional Biology SureSilencing sirna Arrays & shrna Plasmids ChIP-qPCR Assays & Master Mixes CASE Kits, ELISArray Kits
Discovery of RNA Interference Models of development C. elegans and D. melanogaster Expressed genes suppressed in a temporal fashion Biochemical mechanism ascertained Antisense RNA for targeted gene expressed Long RNA causes message degradation Short RNA inhibits translation or message degradation Conserved in all species including mammals Exogenously suppress expression of gene of interest
How Mammalian RNA Interference Works Experimentally in Cultured Mammalian Cells in vitro P mirna (micro RNA) pre-mirna (precursor micro RNA) pri-mirna (primary micro RNA) Transfection P Dicer P RISC sirna (Small Interfering RNA) P P RISC mrna exonucleases Translation Inhibition
RNA Interference Studies Why knock down the expression of a gene? Study the function of a discovered and validated gene of interest Knockdown expression to remove protein activity Perform other assays of cell function to determine missing or changed activities Missing or changed activity attributed to the silenced gene More typically used and historical methods take a long time: Knock-out mice (months) Site-directed mutagenesis (weeks) RNA Interference only take days to perform.
RNA Interference Studies Why knock down the expression of multiple genes? Screen for genes important to a biological process Knock down several genes (or groups of genes) Determine effect on a biological assay Further characterize individual genes (Or subdivide groups & repeat screen) Screen for drug targets Knock down several genes or groups of genes Determine if desired biological process is inhibited Design and develop drugs for genes protein products
RNA Interference Challenges Library Sizes Most sirna libraries offer too many genes at once Too time-consuming or expensive for most researchers Effectiveness & Specificity: Reconciling differences between knockdown efficiencies advertised by companies and observed by researchers Addressing off-target and toxic side-effects insuring phenotypes only due to knocking down gene of interest Applicability: Compatibility to cell-based phenotype assays Appropriate controls for RNAi and phenotype assay
What SureSilencing sirna Arrays Provide Pathway Focused: Knock down 42 key genes in the same biological pathway Reliable and Proven: All sirna sequences are experimentally validated to deliver greater than 70% knockdown and designed to minimize offtarget effects. Ease-of-Use & High-Performance: Simple reverse transfection protocol, convenient plate design for cell-based assays, and high-efficiency transfection reagent make the functional study of pathways accessible to everyone.
What ARE SureSilencing sirna Arrays? RNA interference solution for functional studies on pathways Validated sirna (2 sequences per gene) for 42 pathwayfocused genes plus four replicated RNAi controls in a 96-well cell culture plate with clear bottom and black walls Reverse transfect sirna & directly determine cell phenotype on same plate with colorimetric, luminescent or fluorescent cellbased assays Quickly identify genes involved in a biological process or disease state, the targets of chemical compounds, and the biological impact of your genes of interest in a pathway
SureSilencing sirna Array Applications SureSilencing sirna Arrays are designed to study gene function with cell-based assays. The compatible cell-based assays that you can directly conduct on the sirna Array include: 1. Fluorescence assays for cell viability 2. Chemiluminescence assays for enzyme activity 3. Luciferase reporter assays for pathway activation 4. Colorimetric assays for protein phosphorylation 5. Microscopy for morphology & immunostaining
Layout of the sirna Array Gene-Specific sirna (A1 G12) Duplicate wells of sirna pairs targeting thoroughly researched panel of 42 relevant, pathway-focused genes. Immediately adjacent wells contain replicates for easy analysis of phenotype assay data readout.
sirna Array Control Wells Negative Control sirna: NEG (Wells H1 H4) Compare or normalize the results of phenotype assay to ascertain effects of gene-specific sirna effects on assay Cell But No sirna: NOR (Wells H5 H8) Serves as a mock transfection for phenotype assay normalization
sirna Array Control Wells Transfection Efficiency Control: TEC (Wells H9 H10) Fluorescently labelled negative control sirna monitored by fluorescence microscopy Phenotype Assay Background: NOC (Wells H9 H10) Lack sirna and cells to measure phenotype assay background
SureSilencing sirna Array Protocol Overview Resuspend sirna in Opti-MEM. Add SureFECT in Opti-MEM. Incubate 20 min. Add cells in normal growth medium. Reverse transfect for 24 48 72 h. Check transfection efficiency after 24 h. Perform cell-based phenotype assay. Transient transfection conditions require cells with consistently high transfection efficiencies.
SureSilencing sirna Validation Process Triplicate transfections of negative control sirna and each sirna pair per gene into HeLa, MB-231, HEK 293H, or A549 cells After 48 hours, isolate total RNA Triplicate real-time RT-PCR characterization of the gene of interest (GOI) and housekeeping gene (HKG) for each of the triplicate transfections Use C t to calculate relative gene expression level (percent knockdown)
SureSilencing sirna Array Performance The Knockdown by Each Gene-Specific sirna Pair in Every Array is Experimentally Validated.
SureSilencing sirna Array Applications Accurate Pathway Analysis: SureSilencing sirna Arrays include positive and negative regulators as well as key members of a biological pathway. You can systematically analyze the pathway to identify the relevant genes to your biological research. Functional Analysis of Novel Genes: Determine the genetic and functional relationships of novel genes of interest to genes representative of a biological pathway on a SureSilencing sirna Array. Drug Target, Enhancer, or Inhibitor Validation: SureSilencing sirna Array can also be used to validate drug targets and identify enhancers or inhibitors of a drug s activity.
Drug Target, Enhancer, or Inhibitor Validation Staurosporine is a broad-spectrum kinase inhibitor and a strong apoptosis inducer. Curcumin, the principal curcuminoid in turmeric, exhibits a milder pro-apoptotic activity in cancer cells, but has no cytotoxic effects on healthy cells. Can new drug targets be found to enhance the proapoptotic anti-tumor effects of these compounds? Reverse transfect MDA-MB-231 cells in the Human Apoptosis SureSilencing sirna Array for 48 h. Treat with 0.5 µm staurosporine or curcumin for 16 h. Monitor caspase 3 & 7 activity with cell-based luminescent assay as measure of apoptosis activation
Apoptosis Application Staurosporine Curcumin Knockdown of BCL2L1 and IAP Family Members Stimulates Staurosporine-Induced Apoptosis in Breast Cancer Cells
Accurate Pathway Analysis What factors mediate TNFα stimulation of NFκB Signaling? Reverse transfect HEK-293H cells, containing the Cignal NFκB Pathway Reporter Assay, in the Human NFκB Signaling Pathway sirna Array for 48 h. Treat with 50 ng/ml TNFα for 5 h. Determine relative NFκB activity by luminescence and gene knockdown by real-time RT-PCR.
NFκB Signaling Application RELA but not RELB, and TNF Receptor 1A but not 10A or 10B mediate TNFα-dependent NFκB signaling.
Available Arrays: HOW TO ORDER Human Apoptosis SAH-012A TNF Ligands & Receptors BCL2, IAP, TRAF, and CARD Family Members Death & Death Effector Domain Family Members Caspases & p53 and ATM pathways. Human NFκB Signaling SAH-025A REL, NFκB, and IκB Family Members Extracellular Ligands & Receptors Kinases & Transcription Factors Accessory Reagent: SA-01 SureFECT Reverse Transfection Reagent (For 12 sirna Arrays) Off-the-Shelf Availability
SureSilencing sirna Array Technology Overview Pathway-Focused sirna-based RNA Interference Questions? Contact Technical Support 9 AM 6 PM Eastern M F Telephone: 888-503-3187; Email: support@superarray.net