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Ribonucleic acid (RNA) stands as a fundamental constituent in the architecture of life, a molecule essential for comprehending the intricacies of living organisms. RNA sequencing (RNA-seq) is indispensable for researchers seeking to unravel the functional nuances embedded within RNA. Diverse RNA molecules, each with unique roles in regulating gene expression and epigenetic traits, contribute to the dynamic landscape of biological processes. Among these, small RNA (sRNA), classified as a non-coding RNA (ncRNA), has gained prominence by unraveling complex biological mechanisms.
In this blog, you will learn:
Keywords: sRNA-seq, small RNAs, non-coding RNAs
A distinguishing feature of many types of small RNAs (sRNAs) is their pivotal role in gene silencing, a phenomenon also referred to as RNA silencing. This process is executed through RNA interference (RNAi), where sRNAs actively degrade mRNA, thereby silencing gene expression. The application of sRNA-seq enables researchers to discern the presence of diseases such as breast cancer, Parkinson’s disease, or multiple sclerosis. Additionally, sRNAs contribute to the post-transcriptional regulation of gene expression. Through these multifaceted functions, sRNAs emerge as essential players in the intricate regulatory landscape of cellular processes.
One established method for categorizing non-coding RNAs (ncRNAs) involves consideration of their nucleotide chain length. According to this classification, small RNAs (sRNAs) are characterized as non-coding RNA molecules with a length of fewer than 200 nucleotides. As our understanding of sRNAs has expanded, diverse subtypes of sRNA have been identified, each with unique features and functions:
The key breakthrough in advancing the understanding of small RNAs (sRNAs) was achieved through the implementation of next-generation sequencing (NGS). This technology prioritizes ultra-high throughput, scalability, and rapidity in determining nucleotide sequences of RNA samples. In comparison to preceding methods like microarrays, NGS demonstrates superior capabilities for the detection of novel RNA transcripts, delivering expedited results with heightened specificity and sensitivity.
Sequencing sRNA using NGS is a complex process that unfolds over four stages. Let’s look at each of them in greater detail:
Fig 1. Library construction workflow
The standard pipeline for sRNA-seq analysis, applicable across various sRNA types such as miRNA, piRNA, and siRNA, unfolds as follows:
The table below illustrates the entire pipeline:
Fig 2. Bioinformatics analysis pipeline
Small RNA sequencing (sRNA-Seq) serves as a robust research tool with diverse applications:
The applications outlined above underscore the versatility and utility of sRNA-seq in elucidating functional aspects of small RNA molecules across diverse biological contexts.
One particular use of sRNA-seq in the case of miRNAs we would highlight was performed by Li et al. (2023). The authors used a combination of miRNA sequencing (miRNA-Seq) and bioinformatics analysis to identify miRNAs associated with colorectal cancer and to investigate the role of miRNAs in post-transcriptional control. The authors combined the expression profiles obtained from in-house miRNA-seq, TCGA, GEO, and other array databases from the National Center for Biotechnology Information (NCBI) web server to identify miRNAs associated with colorectal cancer. They then used bioinformatics analysis to predict the target genes of the identified miRNAs and to investigate the functional implications of miRNA binding on gene expression. They also performed luciferase reporter assays to validate the predicted miRNA binding sites in the 3’-UTR of BET1L and other target genes. These techniques allowed the researchers to identify miR-140-3p as a key regulator of BET1L expression and to investigate the functional implications of miRNA binding on gene expression.
Using a single-end strategy of the Illumina NovaSeq sequencing platform, Novogene’s sRNA-seq services deliver comprehensive, reliable results. This NGS method allows Novogene to accurately capture the entire sRNA transcriptome with extreme sensitivity and high resolution in a single analysis. Have all your sRNA bioinformatics requirements met with the help of Novogene’s sRNA-Seq services.
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