Full-Length Transcriptome Analysis of Human Retina-Derived Cell Lines ARPE-19 and Y79 Using the Vector-Capping Method

Abstract

PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina.

Genome‐wide two‐locus interaction analysis identifies multiple epistatic SNP pairs that confer risk of prostate cancer: A cross‐population study

“…chr7:9310980–9331512 is on 7p21, and is at the intergenic region near a noncoding gene RBSG3 (retinoblastoma‐specific gene 3). The sequences of this gene were derived only from retinoblastoma . Bookstein et al .…”

“…The sequences of this gene were derived only from retinoblastoma. 36 Bookstein et al confirmed the existence of retinoblastoma mutations in some human prostate carcinomas. 37 And the retinoblastoma tumor suppressor gene is functionally inactivated in a broad range of both paediatric and adult cancers.…”

High Prevalence of Mutations in theEYSGene in Japanese Patients with Autosomal Recessive Retinitis Pigmentosa

“…Recently, we showed that human retinoblastoma Y79 expressed three variants of the EYS gene, including a long full-length cDNA clone with 7989 bp encoding a short form of EYS with 594 amino acids, but were not able to obtain the full-length cDNA clone encoding the longest form of EYS. 25 Thus, there seem to be many forms of EYS variants, including unknown ones. Given that the short EYS variants contain the N-terminal EGF domains identical to those of the long form, these variants may partially compensate for the loss of the long form of EYS.…”

Peregrine

“…RNA-Seq library preparation methods that provide uniform, full-length sequence coverage of expressed transcripts are particularly useful when characterizing transcription start sites, 31 5′-/3′-untranslated regions, 32 , 33 splicing variants, 34 , 35 operon organization 36 and pathogen transcriptomes 37 - 39 . Thus, in evaluating the performance of Peregrine library preparation it was important to characterize the sequencing coverage provided in terms of its distribution along the length of individual transcripts.…”