Published online 20 June 2005
Methods Online |
High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Program of Molecular and Cellular Biology and Biochemistry Boston, MA 02215, USA 1Center for Advanced Biotechnology 36 Cummington Street, Boston, MA 02215, USA 2Centre for Emerging Infectious Diseases, 2/F, School of Public Health, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital Shatin, New Territories, Hong Kong Special Administrative Region
*To whom correspondence should be addressed. Tel: + 852 2252 8842; Fax: + 852 2635 4977; Email: cmding{at}cuhk.edu.hk
Received April 10, 2005. Revised May 20, 2005. Accepted June 2, 2005.
Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.
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