RIG-1 dsRNA Quantification Kit

Sample

Biological samples

Detection Method

cELISA

Intended Use

This kit is for quantitative measurement of residual dsRNA.

Contents of Kit

RIG-I ATPase Reaction
1. RIG-I enzyme(1 μM) 100 μL
2. RIG-I Reaction Buffer (10×) 0.25 mL
3. 1 mM DTT 20 μL
4. 10 mM ATP 25 μL
5. Standard (UTP, 25 ng/μL) 40 μL
6. Standard (N1-Me-pUTP, 25 ng/μL) 40μL

Measurement of ADP
7. Reagent 1-Assay buffer 0.5 mL
8. Reagent 1-Enzyme(5×) 0.125 mL
9. Reagent 1-0.5M MnCl₂ 25 μL
10. Reagent 2 1.25 mL
11. 384-well Plate 1 plate
12. Instruction manual 1 copy

Storage

1. Dry Ice Shipping. Store the system at -25~- 15°C.
2. Before use, all components should be thawed and mixed thoroughly at room temperature. Once prepared, dispense all reagents into aliquots and store at -25~-15°C. Prepare the RIG-I system and Reagent 1 fresh for each use. Reagent 2 can be placed at room temperature (18–25°C) for 24 hours without significant signal loss.

General Description

Retinoic acid-inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. Structurally, RIG-I is comprised of three main functional domains – Caspase Activation and Recruitment Domain (CARD) for signal transduction, DExD/ H-box RNA helicase domain, and C-terminal domain for RNA binding. The RNA helicase domain is associated with RNA-dependent ATP hydrolysis activities, which is activated upon binding to double-stranded RNA (dsRNA), leading to the hydrolysis of ATP into ADP and inorganic phosphate (Pi). This kit quantifies dsRNA content in samples by measuring ADP production during the reaction between RIG-I and the sample. The assay is performed in a multi-well plate and can detect reactions in volumes as low as 5 μL. The detection involves three steps:
1. RIG-I Reaction: Mix RIG-I enzyme, ATP, and the sample, followed by incubation.
2. ATP Depletion: Add an equal volume of Reagent 1 to deplete residual ATP.
3. ADP Detection: Add Reagent 2 to convert ADP back to ATP, which is then quantified using a luciferase/luciferin reaction. The luminescent signal is measured with a plate reader. dsRNA content is calculated based on a standard curve.