Abstract
BackgroundThe majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use.ResultsHere, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified within a single sample using one-step reverse transcription quantitative PCR. Amplification products obtained with variant specific primer pairs were compared to those obtained with primer pairs common to both variants. The identities of variant specific amplicons were simultaneously verified by melt curve analysis. Independent calculations of the relative incidence of each variant were performed. Since the relative incidences of variants have to add upto 100 %, the method provides an internal control to monitor experimental errors and uniform reverse transcription. The reliability of the method was tested using mixtures of cDNA templates as well as RNA samples from different sources.ConclusionThe method described here, is easy to set up and does not need unrelated reference genes and time consuming, error-prone standard curves. It provides a reliable and precise technique to distinguish small differences of the relative incidence of two splice variants.
Highlights
The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts
Strategy of the method To determine the relative incidence of two splice variants the method of choice should be reliable and as simple as possible
For the sake of simplicity and robustness, we used one-step RT-PCR with gene specific priming of reverse transcription
Summary
The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. Ninety-two to ninety-four percent of the human genes express primary RNA transcripts that undergo alternative splicing [1]. This provides an extensive collection of protein isoforms that display different properties and serve a huge variety of functions [2]. This way, alternative splicing is one of the main mechanisms to expand biological diversity. It is a tightly regulated process and splicing errors cause a number of human diseases [3]. To investigate the significance of splicing and the role of each isoform, a well-founded knowledge of the incidence of alternatively spliced transcripts in a defined cell type or tissue is indispensable
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