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Running the PCR products on an agarose gel, we found that tumor samples of ESCC and some paired non-tumor tissues consistently produced an additional 93 bp fragment in addition to the predicted 201 bp PCR product (Fig. 2B). Direct sequencing of the amplified products further confirmed the identity of the 93 bp band as an alternatively spliced form of SOX2OT, lacking exon 4 of the main transcript (SOX2OT-S1; Accession no: JN711430, GI:379031002). Finding the aforementioned variant encouraged us to search for other potential variants of SOX2OT. Using another specific set of primers (set 2), in which the forward and reverse primers were located on the first and last exons (exon 5) of the gene (Fig. 3A, 3B), another splice variant of SOX2OT lacking exons 3 and 4 was identified by direct sequencing of the PCR products (SOX2OT-S2; Accession no: JN882275, GI:379031003). To examine the expression pattern of the newly discovered variants in ESCC samples, specific primer pairs for SOX2OT splice variants on regions that are distinct for each transcript were used. The results of qRT-PCR revealed that SOX2OT-S1 and SOX2OT-S2 were overexpressed in tumor samples of ESCC, compared to their marginal non-tumor tissue counterparts. Furthermore, overexpression of SOX2OT-S2 in tumor samples, similar to SOX2 and OCT4, was statistically significant (p? the ROC curve analysis, SOX2OT-S1 (AUC?=?0.71, p?=?.0095; Supporting Information Fig. S1D) and SOX2OT-S2 (AUC?=?0.735, p?=?.005; Supporting Information Fig. S1E) seem to be good candidates for discriminating tumor from non-tumor samples. According to the Spearman's rho test, SOX2OT-S1 showed a high correlation to SOX2 (72%, p?=?.01) and a weak correlation to OCT4A (37%, p?=?.05). Similarly, SOX2OT-S2 showed a relatively high correlation to SOX2 (65%, p?=?.015) and OCT4A (59%, p?=?.022). We also examined the expression patterns of the novel variants of SOX2OT (normalized to that of internal control, GAPDH) in NT2 cells by qRT-PCR. SOX2OT-S1 had the least expression level, compared to SOX2OT and SOX2OT-S2 splice variants in the cells. As mentioned above, the SOX2OT-S1 and SOX2OT-S2 variants are expressed in NT2 cells, albeit at lower levels compared to those of OCT4 and SOX2. To examine whether splicing influence the stability of SOX2OT transcripts, we treated NT2 cells with a general transcription inhibitor agent, actinomycin D, and quantified the expression levels of the variants in the treated cells after 0.5, 1.5, 3, 6, and 12 hours. The obtained data revealed that SOX2OT-S2 is the most stable variant, compared to SOX2OT and SOX2-S1 variants (p?