Exon Array Analysis using re-defined probe sets results in reliable identification of alternatively spliced genes in non-small cell lung cancer

Table 1 Validation results of candidate genes.

		Laboratory results
Gene	Alt. splicing hypothesis	RT-PCR	Sequencing	qRT-PCR AdCa	qRT-PCR SCC	Status
ADD3	Cassette exon	4/6	+	+	+	validated
ANTXR1	Alt. transcription start site	N/A	N/A	-	-	devalidated
CLSTN1	Cassette exon	4/6	+	+	-	validated
CTNND1	Cassette exon (5')	-	N/A	+	+	devalidated
	Cassette exon (3')	2/6	+	-	-	devalidated
FN1	Cassette exon	5/5	+	+	+	validated
KIAA1217	Alt. transcription start site	N/A	N/A	+	+	validated
	Intron retention	2/6	+	-	-	devalidated
MYH14	Cassette exon	-	+	-	-	devalidated
MYO18A	Cassette exon	4/6	+	+	-	validated
NCOR2	Alternative 5'-splice site	4/6	+	+	-	validated
NUMB	Cassette exon	5/6	+	+	+	validated
SLK	Cassette exon	5/6	+	+	+	validated
SYNE2	Cassette exon	6/6	+	+	-	validated
TPM1	Mutually exclusive exon (5')	N/A	+	+	+	validated
	Mutually exclusive exon (3')	N/A	+	+	+	validated

A hypothesis of the mode of alternative splicing was formulated based on exon array expression profile and available transcript annotations. Events with alternative start of transcription were also considered, although they are not alternative splicing events by definition. RT-PCR was conducted using six samples; shown here is the number of samples that show an alternative splicing pattern that is in concordance with the exon array results. Exon-exon junctions in RT-PCR products were examined by sequencing. qRT-PCR was performed using transcript variant specific primer pairs for both AdCa and SCC. qRT-PCR results confirmed the exon array results (+) if they showed a significant (paired t-test p ≤ 0.05) or high (SI ≥ 2.0 or SI ≤ 2.0^-1) difference of transcript variant expression that was in agreement with the exon array results. Differential splicing of a gene was considered as validated if at least one of the qRT-PCR results was positive. If available, RT-PCR had to confirm the exon array results in the majority of samples and also sequencing results had to be positive. Detailed result see additional file 11. N/A indicates that the experiment was not performed.

ISSN: 1471-2164