选择性剪接(AS)是真核生物中的普遍现象,识别AS事件仍然具有挑战性。已经开发了几种方法来识别AS事件,如表达序列标签(EST),微阵列和RNA-seq。然而,EST在识别低丰度基因方面有局限性,而微阵列和RNA-seq是高通量技术,并且需要基于PCR的技术进行验证。为了克服EST的局限性和高通量技术的缺点,我们建立了一种识别AS事件的方法,尤其是低丰度基因,通过逆转录(RT)用基因特异性引物(GSP)进行PCR,然后进行巢式PCR。此过程包括两个主要步骤:1)使用GSPs扩增只要特定的基因片段和2)多轮巢式PCR筛选AS并确认未知的剪接变体。使用这种方法,我们成功鉴定出三个新的剪接变体,即,GenBank登录号用于bdnf基因的HM623886(GenBankGeneID:12064),GenBank登录号Trkc基因的JF417977(GenBankGeneID:18213)和GenBank登录号。glb-18基因的HM623888(GenBankGeneID:172485)。除了它的可靠性和简单性,该方法还具有成本效益和劳动密集型。总之,我们开发了一种使用基因特异性引物的RT-巢式PCR方法,以有效地鉴定已知和新的AS变体。该方法克服了用于检测稀有转录物的现有方法的限制。通过发现新的亚型,尤其是低丰度基因,这项技术可以帮助研究疾病中的异常剪接。未来的研究可以应用这种方法来发现与癌症有关的AS变异,神经变性,和其他剪接相关疾病。
Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.