Reverse protection assay: a tool to analyze transcriptional rates from individual promoters

Today's molecular biology employs several methods to determine the steady-state levels of RNAs, such as Northern- and dot-blot analysis, ribonuclease protection assay (RPA), reverse transcription-PCR (RT-PCR), and quantitative real-time PCR. These methods differ in their sensitivity and information content. For example, while RT-PCR allows quantification of RNA molecules of different lengths, RPA permits determination of the relative amount of transcripts with distinct 5'- and/or 3'-ends [1]. In general, RPA is based on hybridization of a labeled, single-stranded antisense RNA probe to the target RNA. Subsequent incubation with an RNases mix degrades those RNA molecules that do not form double-stranded hybrids. Final inactivation of RNases and precipitation of the protected RNA hybrids is followed by electrophoretic analyses, which reveals the presence, size, and relative level of RNA that was protected by the antisense probe [1]. The RPA method has been modified to serve for different tasks, such as measuring the radioactive signals by scintillation counting [RiPPA method; [2]], and replacing radiolabeled with biotinylated probes [3]. Adding constitutively transcribed or in vitro synthesized RNA as an internal standard during RNA isolation allows quantification of the RNA analyzed by RPA [4,5].However, the overall RNA content reflects a balance between the synthesis and degradation of transcripts [6]. The aforementioned methods only identify RNA steady-state levels. To directly evaluate the rate of individual gene transcription, run-on transcription is utilized: transcripts are labeled by adding radio-nucleotides during a brief time of incubation and subsequently analyzed by dot-blot hybridization [1]. This technique applies to all genetic compartments such as bacterial cells, nuclei, mitochondria, and chloroplasts. However, this method also has some limitations. The double-stranded probes usually used for dot-blots in run-on assays are able to hybridize with both