RPAD (RNase R treatment, polyadenylation, and poly(A)+ RNA depletion) method to isolate highly pure circular RNA.
Methods
; 155: 41-48, 2019 02 15.
Article
em En
| MEDLINE
| ID: mdl-30391514
ABSTRACT
Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Poli A
/
RNA
/
RNA Mensageiro
/
Análise de Sequência de RNA
/
Biologia Computacional
Limite:
Humans
Idioma:
En
Revista:
Methods
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Estados Unidos