Dataset Information

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

ABSTRACT: Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3' UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3' RACE. Expanded 3' RACE can detect unusual 3' UTRs, including gene fusions within the 3' UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

SUBMITTER: Masamha CP

PROVIDER: S-EPMC5933262 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

Masamha Chioniso Patience CP Todd Zachery Z

Journal of visualized experiments : JoVE 20180328 133

Maturation of eukaryotic mRNAs involves 3' end formation, which involves the addition of a poly(A) tail. In order to map the 3' end of a gene, the traditional method of choice is 3' rapid amplification of cDNA ends (3' RACE). Protocols for 3' RACE require the careful design and selection of nested primers within the 3' untranslated region (3' UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3' UTR and sequences within the open ...[more]

PMID: 29658915

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Project description:BackgroundAlternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by ERBB2 (HER-2/neu) oncogene expression.ResultsThe human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of ERBB2-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of ERBB2. The relative expression balance between AS variants from 3 genes was differentially modulated by ERBB2 in this model system.ConclusionsIn this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by ERBB2-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.

Dataset Information

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

Publications

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer.

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