Simultaneous Quantification of Multiple Alternatively Spliced mRNA Transcripts Using Droplet Digital PCR.
Methods Mol Biol
; 1768: 387-400, 2018.
Article
em En
| MEDLINE
| ID: mdl-29717455
ABSTRACT
Currently there is no sensitive, precise, and reproducible method to quantitate alternative splicing of mRNA transcripts. Droplet digital™ PCR (ddPCR™) analysis allows for accurate digital counting for quantification of gene expression. Human telomerase reverse transcriptase (hTERT) is one of the essential components required for telomerase activity and for the maintenance of telomeres. Several alternatively spliced forms of hTERT mRNA in human primary and tumor cells have been reported in the literature. Using one pair of primers and two probes for hTERT, four alternatively spliced forms of hTERT (α-/ß+, α+/ß- single deletions, α-/ß- double deletion, and nondeletion α+/ß+) were accurately quantified through a novel analysis method via data collected from a single ddPCR reaction. In this chapter, we describe this ddPCR method that enables direct quantitative comparison of four alternatively spliced forms of the hTERT messenger RNA without the need for internal standards or multiple pairs of primers specific for each variant, eliminating the technical variation due to differential PCR amplification efficiency for different amplicons and the challenges of quantification using standard curves. This simple and straightforward method should have general utility for quantifying alternatively spliced gene transcripts.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA Mensageiro
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Processamento Alternativo
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Telomerase
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article