MilliporeSigma
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11468120910

Roche

Reverse Transcriptase Assay, colorimetric

suitable for enzyme immunoassay, kit of 1 (14 components), sufficient for ≤200 tests

Synonym(s):
RT-PCR

Quality Level

usage

sufficient for ≤200 tests

packaging

kit of 1 (14 components)

manufacturer/tradename

Roche

technique(s)

enzyme immunoassay: suitable

shipped in

dry ice

storage temp.

−20°C

General description

Colorimetric enzyme immunoassay for the quantitative determination of retroviral reverse transcriptase activity by incorporation of digoxigenin- and biotin-labeled dUTP into DNA.

Application

The Reverse Transcriptase Assay is designed for the quantitative determination of RT activity in cell culture and other biological samples. The assay is used to determine the propagation of retroviruses in retrovirus-infected mammalian cells in culture. The assay is also used for in vitro screening for RT inhibitors.

Packaging

1 kit containing 14 components.

Specifications

Assays for reverse transcriptase (RT) activity have been broadly applied for testing retroviral propagation in vitro. RT is required for early proviral DNA synthesis, and is thus a prime target for anti-retroviral therapy, for example, in the case of acquired immunodeficiency syndrome (AIDS). Reverse transcriptase generally uses RNA, which is complexed with various primers, as a template for DNA synthesis. Classically, for the detection or quantification of RT activity, the amount of incorporated radioactively labeled nucleotides is measured.
Assay time: Approximately 4 hours (for an enzyme reaction of 1 hour), approximately 18 hours (for an enzyme reaction of 15 hours).
Sample material: Cell culture supernatant and other biological samples, RT inhibitors.
Sensitivity: The Reverse Transcriptase Assay detects 20pg HIV-1 reverse transcriptase in a 1-hour reaction. One pg can be detected in an overnight reaction.
The assay detects the activity of natural or recombinant retroviral reverse transcriptase, including that of HIV-1, HIV-2, SIV-1, AMV, and M-MulV.

Principle

The Reverse Transcriptase Assay, colorimetric, takes advantage of the ability of reverse transcriptase to synthesize DNA using the hybrid poly (A) x oligo (dT)15 as a template and primer. It avoids the use of [3H]- or [32P]-labeled nucleotides that are employed in standard RT assays. In place of radiolabeled nucleotides, digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into the same DNA molecule by the RT activity. A template/primer hybrid is supplied, but the flexibility of the assay allows the use of a template of choice (e.g., a viral template). The detection and quantification of the synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol: biotin-labeled DNA binds to the surface of streptavidin-coated microplate modules. In the next step, an antibody to digoxigenin, conjugated to peroxidase (anti-DIG- POD), is added and bound to the digoxigenin-labeled nucleotides (Licensed by Institute Pasteur). In the final step, the peroxidase substrate ABTS is added. The peroxidase enzyme catalyzes the cleavage of the substrate to produce a colored reaction product. The absorbance of the samples is determined using a microplate (ELISA) reader, and is directly correlated to the level of RT activity in the sample.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

Product No.
Description

  • HIV-1 Reverse Transcriptase

  • Incubation Buffer

  • Nucleotides

  • Template/Primer Hybrid

  • Lysis Buffer

  • Anti-Digoxigenin-POD antibody

  • Washing Buffer Concentrate 10x concentrated

  • Conjugate Dilution Buffer

  • Substrate Buffer

  • ABTS Substrate

  • Substrate Enhancer

  • Microplate Modules, precoated with streptavidin

  • One Strip Frame (for 8-well microplate modules)

  • Cover Foils for Microplates

See All (14)

Pictograms

CorrosionExclamation mark

Signal Word

Danger

Hazard Statements

Hazard Classifications

Aquatic Chronic 3 - Eye Dam. 1 - Skin Sens. 1

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis

Enter Lot Number to search for Certificate of Analysis (COA).

Certificate of Origin

Enter Lot Number to search for Certificate of Origin (COO).

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