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Nucleic Acids Research, 2001, Vol. 29, No. 13 e68
© 2001 Oxford University Press

Quantification of splice variants using real-time PCR

Ina I. Vandenbroucke, Jo Vandesompele, Anne De Paepe and Ludwine Messiaen* Department of Medical Genetics, University Hospital Ghent-OK5, De Pintelaan 185, 9000 Ghent, Belgium

A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants has been laborious and difficult due to the intrinsic limitations of conventional methods. The many advantages of real-time PCR have made this technique attractive to study its application in quantification of splice isoforms. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. An overview of three different possibilities for detecting alternative transcripts is given. We propose the use of a boundary-spanning primer to quantify isoforms that differ greatly in abundance. We describe here a novel method for creating a reliable standard curve using one plasmid containing both alternative transcripts. In addition, we validate the use of an absolute standard curve based on a dilution series of fluorometrically quantified PCR products.

* To whom correspondence should be addressed. Tel: +32 9 240 2478; Fax: +32 9 240 4970; Email: ludwine.messiaen@rug.ac.be


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