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

The SureSilencing siRNA Arrays may be used for:

• 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 Arrays can also be used to validate drug targets and identify enhancers or inhibitors of a drug's activity.

Accurate Pathway Analysis: Human NFκB Signaling
TNF Receptor 1A and RELA are Required for TNFα Activation of NFκB Signaling

Figure 1: TNF Receptor 1A and RELA Are Required for TNFα Activation of NFκB Signaling.

HEK-293H cells, containing the Cignal™ NFκB Pathway Reporter Assay, were reverse transfected in the Human NFκB Signaling Pathway siRNA Array for 48 h. After treatment with 50 ng/ml TNFα for 5 h, relative NFκB activity was analyzed by luminescence (red bars), and gene knockdown was also determined by real-time RT-PCR (blue symbols).

RELA knockdown abolishes the NFκB reporter activity seen upon TNFα stimulation, whereas RELB knockdown has a much reduced effect. Only knockdown of TNF receptor 1A, and not 10A or 10B, significantly inhibits TNFα stimulation of reporter activity. Therefore, RELA but not RELB, and TNF Receptor 1A but not 10A or 10B mediate TNFα-dependent NFκB signaling.

SureSilencing siRNA Arrays can systematically analyze pathways to identify the relevant genes to your biological research.

Target, Enhancer, or Inhibitor Validation & Identification: Human Apoptosis
Knockdown of BCL2L1 and IAP Family Members Stimulates Staurosporine-Induced Apoptosis in Breast Cancer Cells

Figure 2: Knockdown of BCL2L1 and IAP Family Members Stimulates Staurosporine-Induced Apoptosis in Breast Cancer Cells

MDA-MB-231 cells were reverse transfected in the Human Apoptosis SureSilencing siRNA Array for 48 h. Upon treatment with 0.5 µM staurosporine or curcumin for 16 h, caspase 3 and 7 activity was then measured using a cell-based luminescence assay. 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.

Knockdown of a BCL2 family member, BCL2L1, enhances the caspase activation induced by STS 4.4-fold. Three IAP family members, BIRC2, BIRC4 and BIRC5, also significantly augment the effect of STS by 3.4-, 2.5-, and 4.2-fold, respectively. Only knockdown of BCL2L1 significantly enhances the caspase activation effect of curcumin (5-fold). New compounds inhibiting the activity of the corresponding gene products may then improve the ability of STS or curcumin to induce apoptosis.

SureSilencing siRNA Arrays can be used to validate drug targets and identify enhancers or inhibitors of a drug's activity.