Reverse transcriptase is an enzyme that is particular to retroviruses which include HIV as well as a number of murine and avian retroviruses. Since this enzyme is unique to the retrovirus family, detection of reverse transcriptase enzymatic activity is a suitable measure to monitor both rates of infections or as a screening tool for identification of reverse transcriptase inhibitors.
Reverse transcriptase generates DNA from RNA. In this reaction, long RNA-DNA heteroduplexes are formed. Molecular Probes EnzCheck Reverse Transcriptase (RT) Assay Kit takes advantage of this RT product by using their PicoGreen dsDNA quanitation reagent. Picogreen detects dsDNA or RNA-DNA heteroduplexes over single stranded nucleic acids or free nucleotides. The assay setup is simple. The kit contains the Picogreen dsDNA quantitation reagent, lambda DNA standard, poly(A) ribonucleotide template, oligo d(t) primer, and all necessary buffers. In a 96 well configured format, you first incubate the template and the primer and allow them to anneal. The template/primer mixture is then diluted in a polymerization buffer, and the reaction mixture is aliquoted into a 96 well plate. Samples are then prepared in an appropriate buffer, and can inlcude supernatants, serum samples, or purified RT. A standard curve is generated using the provided lambda DNA. The reaction is allowed to proceed at 25 degrees for 10-60 minutes. The reactions are then stopped by the addition of EDTA. Detection of the RNA-DNA duplex is made by incubating with the PicoGreen solution for 5 minutes. A microplate reader capable of exciting fluorescein (excitation 480 nm, emission 520 nm) is used to measure the amount of labeled RNA-DNA. In a side by side comparison of in vitro modeled HIV infection, we found that the Enzcheck assay was comparable to the traditional radioactive assay in both linearity of signal and range of detection.
The assay has many advantages over traditional RT activity assays in that no radioactivity is required and the time of assay termination is under 2 hours. Standard RT activity assays required radiolabeled phosphate for detection of incorporated nucleotides into the RNA-DNA heteroduplex, followed by spotting of radio-containing material either into scintillation counting tubes or onto phosphocellulose paper and exposed to film for 12-24 hour periods. The subsequent scintillation counts or scanned in densitometry from exposed film would have to be analyzed, a process that could take several days. This assay greatly reduces the time from days to just a couple of hours. The lack of radioactivity is also beneficial when dealing with HIV samples since it becomes a biological and radiological hazard that requires specialized equipment just for HIV related material. The assay is a highly sensitive fluorescent based assay that greatly simplifies the detection of RT activity. One of the drawbacks to the assay is that if you use biological samples such as sera samples, there could be components in the sera that are reactive with the PicoGreen or diminish the fluorescent properties. These components can include elevated levels of salts, such as sodium chloride, and proteins such as bovine serum albumin and immunoglobulins. Detergents such as SDS (1%) or triton-X (0.1%) do not seem to greatly affect fluorescent properties in cases where investigators want to lyse infected cells.
Overall, the Molecular Probes EnzCheck Reverse Transcriptase assay kit is a convenient, simple to use, all in one, RT activity assay that was designed with scalability in mind. I would highly recommend this assay to anyone who has had to perform RT activity assays the conventional way.
Omar Perez, Ph.D.
Department of Microbiology & Immunology
Center for Clinical Science Research