PDF(Pubmed)
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.
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.
摘要：
背景：RNA测序（RNA-seq）广泛用于基因表达谱分析和定量。定量RNA测序通常需要细胞计数和掺入，这并不总是适用于许多样品。这里,我们提出了一种新的定量RNA测序方法，不依赖于刺入或细胞计数，命名为siqRNA-seq，它可以通过利用gDNA作为内部对照来定量描述基因表达。单链文库制备用于siqRNA-seq谱gDNA和cDNA，效率相等。
结果：为了量化mRNA表达水平，用于总核酸的siqRNA-seq构建文库以建立用于表达定量的模型。与相对定量RNA-seq相比，siqRNA-seq在技术上是可靠的，并且对于表达谱分析是可重复的，但也可以对gDNA的读数进行测序，这可以用作精确表达定量的内部参考。应用siqRNA-seq研究放线菌素D对HEK293T细胞基因表达的影响,我们展示了siqRNA-seq在准确识别具有不同全局mRNA水平的样品之间的差异表达基因方面的优势。此外，我们使用siqRNA-seq分析了影响ActD调节的基因表达下降趋势的因素，发现与没有m6A修饰的mRNA相比，具有m6A修饰的mRNA表现出更快的衰减速率。此外,将该技术应用于7种肿瘤细胞系的定量分析，揭示了肿瘤细胞系中总mRNA表达的高度多样性。
结论：总的来说，siqRNA-seq是一种刺入式独立定量RNA测序方法，它创造性地使用gDNA作为内部参考来绝对量化基因表达。我们认为siqRNA-seq提供了一种方便且通用的方法来定量描述各种样品中的mRNA景观。
结果：为了量化mRNA表达水平，用于总核酸的siqRNA-seq构建文库以建立用于表达定量的模型。与相对定量RNA-seq相比，siqRNA-seq在技术上是可靠的，并且对于表达谱分析是可重复的，但也可以对gDNA的读数进行测序，这可以用作精确表达定量的内部参考。应用siqRNA-seq研究放线菌素D对HEK293T细胞基因表达的影响,我们展示了siqRNA-seq在准确识别具有不同全局mRNA水平的样品之间的差异表达基因方面的优势。此外，我们使用siqRNA-seq分析了影响ActD调节的基因表达下降趋势的因素，发现与没有m6A修饰的mRNA相比，具有m6A修饰的mRNA表现出更快的衰减速率。此外,将该技术应用于7种肿瘤细胞系的定量分析，揭示了肿瘤细胞系中总mRNA表达的高度多样性。
结论：总的来说，siqRNA-seq是一种刺入式独立定量RNA测序方法，它创造性地使用gDNA作为内部参考来绝对量化基因表达。我们认为siqRNA-seq提供了一种方便且通用的方法来定量描述各种样品中的mRNA景观。
