EnzChek™ Reverse Transcriptase Assay Kit (Invitrogen™)

The EnzChek Reverse Transcriptase Assay Kit is a convenient, efficient, and inexpensive assay for measuring reverse transcriptase activity and screening reverse transcriptase inhibitors. In less than an hour, samples can be read in a fluorometer or microplate reader with filter sets appropriate for fluorescein (FITC), GFP, or Alexa Fluor 488.

Features of the EnzChek Reverse Transcriptase Assay Kit include:
• Detect as little as 0.02 units of HIV-1 reverse transcriptase
• Large dynamic assay range, detect up to a 50-fold linear range
• Simple to use assay, amenable for automated high-throughput screening applications

The EnzChek Reverse Transcriptase Assay Kit uses the PicoGreen reagent, which preferentially detects dsDNA or RNA-DNA heteroduplexes over single-stranded nucleic acids or free nucleotides. In this assay, the reverse transcriptase activity in a biological sample generates long RNA-DNA heteroduplexes from a mixture of a long poly(A) template, an oligo-dT primer, and dTTP. The RNA-DNA heteroduplexes formed are then detected by the PicoGreen reagent.

Reverse transcriptase is an enzyme that functions as an RNA-dependent DNA-polymerase to synthesize DNA from an RNA template. Retroviruses such as HIV use reverse transcriptase activity to make a DNA copy of their viral RNA to insert into a host cell genome. Reverse transcriptase enzymes are also used in various cell functions, such as the enzyme telomerase, which maintains telomere length and chromosome stability and has a role in cancer and aging. In molecular biology, reverse transcriptase function is utilized to generate complementary DNA (cDNA) libraries from mRNA and quantify mRNA expression through amplification in RT-PCR.

The EnzChek Reverse Transcriptase Assay Kit provides a simple method to determine reverse transcriptase activity that is useful in the study of retroviruses and screening reverse transcriptase inhibitors, which can function as therapeutic drugs to target retroviral diseases. Because this method is faster and less expensive than standard isotopic assays or immunoassays, it is suitable for testing large numbers of biological samples, enabling automated high-throughput screening of reverse transcriptase inhibitors.