Comparison of methods for quantification of subtle splice variants.

Alternative splicing is capable of generating multiple mRNA variants from a single gene and is hence a key mediator of molecular diversity generated at the transcript level. Consequently, delivering quantitative information on the fractions of splice variants is essential for the understanding of their biological roles. Here we compare techniques for subtle splice variant quantification that are able to resolve length differences as small as one nucleotide: PAGE with ethidium-bromide densitometry, pyrosequencing, and CE-LIF. We give comprehensive descriptions of assay designs and calibration procedures and present an evaluation of these methods in terms of accuracy, reproducibility and applicability. We also examined template concentrations and reverse transcription-coupled PCR conditions as potential cause of biased results as they were observed for extreme low template concentrations and/or PCR amplicons with size differences of 195 nt. As proof of concept, we determine the splice ratios of variants differing by 3 and 12 nt in five human tissues. We demonstrate that CE-LIF is the most precise and also the most labor- and time-efficient method.