Detection and measurement of alternative splicing using splicing-sensitive microarrays.
Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.
Srinivasan
Karpagam
K
Department of Molecular, Cell, and Developmental Biology, Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
Shiue
Lily
L
Hayes
Justin D
JD
Centers
Ross
R
Fitzwater
Sean
S
Loewen
Rebecca
R
Edmondson
Lillian R
LR
Bryant
Jessica
J
Smith
Michael
M
Rommelfanger
Claire
C
Welch
Valerie
V
Clark
Tyson A
TA
Sugnet
Charles W
CW
Howe
Kenneth J
KJ
Mandel-Gutfreund
Yael
Y
Ares
Manuel
M
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D034285:Dynamin II / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008954:Models, Biological; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D022821:RNA Splice Sites; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid
D022821:RNA Splice Sites; D034285:Dynamin II