Profiling alternative splicing on fiber-optic arrays.
The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10-100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.
Yeakley
Joanne M
JM
Department of Cellular and Molecular Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Fan
Jian-Bing
JB
Doucet
Dennis
D
Luo
Lin
L
Wickham
Eliza
E
Ye
Zhen
Z
Chee
Mark S
MS
Fu
Xiang-Dong
XD
D017398:Alternative Splicing / Q000235:genetics*; D002460:Cell Line; D005336:Fiber Optic Technology / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D055100:Optical Fibers; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D012333:RNA, Messenger