AQRNA-seq for Quantifying Small RNAs.
J Vis Exp
; (204)2024 Feb 02.
Article
em En
| MEDLINE
| ID: mdl-38372376
ABSTRACT
AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Revista:
J Vis Exp
Ano de publicação:
2024
Tipo de documento:
Article