Detection of alternatively spliced transcripts in leukemia cell lines by minisequencing on microarrays.
BACKGROUND
Recent genome-wide expression studies suggest that approximately 80% of the 25,000 human genes undergo alternative splicing. Alternative splicing may be associated with human diseases, particularly with cancer, but the molecular disease mechanisms are poorly understood. Convenient, novel methods for multiplexed detection of alternatively spliced transcripts are needed.


METHODS
We devised a new approach for detecting splice variants based on a tag-microarray minisequencing system, originally developed for genotyping single-nucleotide polymorphisms. We established the system for multiplexed detection of 61 alternatively spliced transcripts in a panel of 19 cancer-related genes and used it to dissect the splicing patterns in cancer and endothelial cells.


RESULTS
Our microarray system detected 82% of the splice variants screened for, including both simple and complex splice variants, in at least 1 of the leukemia cell types analyzed. The intraassay CV values for our method ranged from 0.01 to 0.34 (mean, 0.13) for 5 replicate measurements. Our system allowed semiquantitative comparison of the splicing patterns between the cell lines. Similar, but not identical, patterns of alternative splicing were observed among the leukemia cell lines. Size analysis of the PCR products subjected to the tag-array minisequencing system and real-time PCR with exon-junction probes verified the results from the microarray system.


CONCLUSIONS
The microarray-based method is a robust and easily accessible tool for parallel detection of alternatively spliced transcripts of multiple genes. It can be used for studying alternative splicing in cancer progression and for following up drug treatment, and it may be a useful tool in clinical diagnostics for cancer and other disorders.
Milani
Lili
L
Molecular Medicine, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
Fredriksson
Mona
M
Syvänen
Ann-Christine
AC
D017398:Alternative Splicing / Q000235:genetics*; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D018076:DNA, Complementary