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siqRNA-seq is a spike-in-independent technique for quantitative mapping of mRNA landscape.
Wang, Zhenzhen; Tao, Kehan; Ji, Jiaojiao; Sun, Changbin; Xu, Wei.
Afiliação
  • Wang Z; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China.
  • Tao K; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China.
  • Ji J; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China.
  • Sun C; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China. sunchangbin@caas.cn.
  • Xu W; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Livestock and Poultry Multi-omics of MARA, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518120, China. xuwei01@caas.cn.
BMC Genomics ; 25(1): 743, 2024 Jul 30.
Article em En | MEDLINE | ID: mdl-39080556
ABSTRACT

BACKGROUND:

RNA sequencing (RNA-seq) is widely used for gene expression profiling and quantification. Quantitative RNA sequencing usually requires cell counting and spike-in, which is not always applicable to many samples. Here, we present a novel quantitative RNA sequencing method independent of spike-ins or cell counting, named siqRNA-seq, which can be used to quantitatively profile gene expression by utilizing gDNA as an internal control. Single-stranded library preparation used in siqRNA-seq profiles gDNA and cDNA with equal efficiency.

RESULTS:

To quantify mRNA expression levels, siqRNA-seq constructs libraries for total nucleic acid to establish a model for expression quantification. Compared to Relative Quantification RNA-seq, siqRNA-seq is technically reliable and reproducible for expression profiling but also can sequence reads from gDNA which can be used as an internal reference for accurate expression quantification. Applying siqRNA-seq to investigate the effects of actinomycin D on gene expression in HEK293T cells, we show the advantages of siqRNA-seq in accurately identifying differentially expressed genes between samples with distinct global mRNA levels. Furthermore, we analyzed factors influencing the downward trend of gene expression regulated by ActD using siqRNA-seq and found that mRNA with m6A modification exhibited a faster decay rate compared to mRNA without m6A modification. Additionally, applying this technique to the quantitative analysis of seven tumor cell lines revealed a high degree of diversity in total mRNA expression among tumor cell lines.

CONCLUSIONS:

Collectively, siqRNA-seq is a spike-in independent quantitative RNA sequencing method, which creatively uses gDNA as an internal reference to absolutely quantify gene expression. We consider that siqRNA-seq provides a convenient and versatile method to quantitatively profile the mRNA landscape in various samples.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: RNA Mensageiro / Análise de Sequência de RNA Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: RNA Mensageiro / Análise de Sequência de RNA Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article