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dsRNA Detection Kit


Category

Kits


Art.No.:

Refer to below


Product Name:

dsRNA Detection Kit


Purity

None

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Technical Parameter

Technical Parameter

Product description

The dsRNA detection kit (Cat. No.: DS0001) adopts the double-antibody sandwich method principle and couples the biotin-streptavidin system to quantitatively detect the double-stranded RNA (dsRNA) content in the sample. The detected dsRNA has nothing to do with its nucleic acid sequence and is 60bp and above in length.The microwells of the microplate are coated with anti-dsRNA antibodies, and the samples are added and then incubated and washed. Then add biotinylated detection antibody and incubate to form an antibody-antigen-antibody complex. After washing again, horseradish peroxidase (HRP)-labeled streptavidin (SA) was added. After thorough washing, the substrate TMB is added to develop color. TMB is converted into blue under the catalysis of peroxidase, and is converted into the final yellow after acid termination. The depth of color is positively correlated with the dsRNA content in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm, and calculate the dsRNA concentration in the sample based on the standard curve.

 

Product components

No.

component

specification

DS0001-A

biotinylated detection antibody (100×)

120μL

DS0001-B

HRP-Streptavidin (100×)

120μL

DS0001-C

diluent

30mL

DS0001-D

TMB substrate solution

12mL

DS0001-E

stop solution

6mL

DS0001-F

Concentrated washing buffer (20×)

40mL

DS0001-G

UTP dsRNA standard (5ng/μL)

10μL

DS0001-H

pUTP dsRNA standard (5ng/μL)

10μL

DS0001-I

N1-Me-pUTP dsRNA standard(5ng/μL)

10μL

DS0001-J

5-OMe-UTP dsRNA standard(5ng/μL)

10μL

DS0001-K

STE buffer

50mL

DS0001-L

ELISA Microplate

8×12-well

DS0001-M

Plate sealer

4 pieces

 

Preparation before experiment

1. Bring all kit components to room temperature (18-25℃).
2. 20× concentrated washing buffer is diluted with purified water at 1:19 volume ratio into washing working buffer.

3. Centrifuge the antibody tubes, HRP- Streptavidin tubes and standard tubes at 1000 rpm for 30 s before use to avoid reagent residue on the tube walls and caps.

4. Dilute biotinylated detection antibody (100×) and HRP-Streptavidin (100×) 100 times with diluent.

5. UTP, pUTP dsRNA standards were diluted to 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0 pg/μL with STE buffer.

N1-Me-pUTP dsRNA standards were diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0 pg/μL with STE buffer.

5-OMe-UTP dsRNA standards were diluted to 8, 4, 2, 1, 0.5, 0.25, 0.125, 0 pg/μL with STE buffer.

 

Experiment protocol
1. Take out the required plate strips from the aluminum foil bag after equilibration at room temperature. Remaining plate strips not used in this assay should cover with plate sealers and stored under 2~8℃ in the ziplock bag. 

2. Set up standard and sample wells. Add 100 μL of standards of different concentrations into the standard well, and add 100 μL of the sample to be tested into the sample well.

3.Cover with the plate sealer. Incubate for 60 min at room temperature with shaking at 500rpm.

4. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250 μL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, repeat washing the plate 4 times

5. Add 100 μL of biotinylated detection antibody at the working concentration to each well of the standard and sample wells, seal the reaction wells with a sealing film, and react with a shaking plate (500 rpm) at room temperature for 60 minutes.

6. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250 μL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 4 times.

7. Add 100 μL of HRP-streptavidin at the working concentration to each well of the standard and sample wells, seal the reaction wells with a sealing film, and react with a shaking plate (500 rpm) at room temperature for 30 minutes.

8. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250 μL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 4 times.

9. Add 100 μL of single-component substrate chromogenic solution to each well, seal the reaction well with a sealing film, and let stand at room temperature for 30 minutes in the dark.

10. Add 50 μL of stop solution to each well, perform detection immediately, and set the wavelength of the microplate reader at 450nm (it is recommended to use dual wavelength 450nm/650nm).

 

 

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