High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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.
McCullough
Ron M
RM
Program of Molecular and Cellular Biology and Biochemistry, Boston, MA 02215, USA.
Cantor
Charles R
CR
Ding
Chunming
C
D000185:Actinin / Q000096:biosynthesis / Q000235:genetics; D017398:Alternative Splicing; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D005091:Exons; D006801:Humans; D008840:Microfilament Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
C111453:ACTN4 protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D008840:Microfilament Proteins; D009692:Nucleic Acid Heteroduplexes; D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; D000185:Actinin