Quan-T-RT™ contains the following components: Annealed primer/template bound to streptavidin-coated SPA beads, assay buffer, stop solution, [3H]TTP (3.7 MBq, 100 µCi) and instructions for use.
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The Quan-T-RT assay system has been optimized for the detection of reverse transcriptase in tissue culture supernatants. The Quan-T-RT™ kit is a reverse transcriptase enzyme assay which makes use of the scintillation proximity assay (SPA) principle. In an aqueous environment, relatively weak β-emitters, notably [3H] and [125I] (Auger electrons), need to be close to scintillant molecules in order to produce light, otherwise the energy is dissipated and lost in the solvent. PerkinElmer has used this concept to develop a range of homogeneous assays by coupling specific molecules on to fluomicrospheres (beads containing scintillant). The Quan-T-RT assay uses a DNA/RNA primer/template, bound to SPA beads via a biotin/streptavidin linkage. No use of liquid scintillant is required —the scintillant is incorporated chemically within the microsphere —and no washing steps are required to eliminate unincorporated radionucleotide from the assay, reducing both the time required for the assay and the potential risks to workers in handling viral samples.
The primer DNA, containing biotin at its 5’ end, is a 16mer oligo(T) which has been annealed to a poly(rA) template, approximately 300 bases in length. The primer/template is bound to a streptavidin-coated SPA bead. Incorporation of [3H]TTP and TTP by reverse transcription, results in extension of the primer. The incorporated tritiated nucleotides are able to stimulate the scintillant and produce a signal. Unincorporated, tritiated nucleotides, free in solution, are essentially unable to stimulate the scintillant and therefore produce no signal. Recombinant HIV-reverse transcriptase (rHIV-RT) can be used as a positive control or for creating a standard curve.
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SPA reagents and CytoStar-T™ microplates let you see what you can’t see with any other technology, so your research becomes more productive. SPA from PerkinElmer. The advantages become even clearer at www.perkinelmer.com/spa.
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