MicroRNAs (miRNAs) and Next-Generation Sequencing (NGS) are powerful tools in molecular biology. MiRNAs are tiny regulators of gene expression, while NGS rapidly sequences genetic material. Combining these technologies provides insights into biological processes and diseases, allowing researchers to explore gene regulation with high precision.
Understanding MicroRNAs
MicroRNAs are small, non-coding RNA molecules, typically 20-25 nucleotides in length. They do not code for proteins themselves but act as regulators of gene expression. MiRNAs achieve this by binding to messenger RNA (mRNA) molecules, primarily in the cell cytoplasm. This binding can either inhibit protein production from the mRNA or promote its degradation.
It is estimated that miRNAs regulate the expression of over 60% of human protein-coding genes. MiRNAs are involved in a wide array of cellular processes, including cell proliferation, differentiation, and programmed cell death (apoptosis). Their involvement extends to various aspects of health and disease, with dysregulation of miRNAs implicated in conditions like cancer, cardiovascular disease, and neurological disorders.
to Next-Generation Sequencing
Next-Generation Sequencing (NGS) refers to technologies that enable the rapid and simultaneous sequencing of millions of DNA or RNA molecules. This capability allows for the generation of vast amounts of genetic information quickly and cost-effectively. NGS provides ultra-high throughput and scalability, transforming the field of genomics.
NGS can sequence entire genomes or specific regions of DNA or RNA, providing detailed insights into genetic variations and gene activity. For example, Illumina NGS systems can produce data ranging from 300 kilobases to multiple terabases in a single run. This technology has impacted genomics and molecular biology, enabling researchers to study biological systems in detail.
Applying NGS for MicroRNA Analysis
NGS is specifically utilized to study microRNAs through a multi-step workflow that begins with sample preparation. High-quality RNA is extracted from various biological specimens, including tissues, cells, and biofluids, with careful attention to preserving small RNA species. Following extraction, the RNA quality and quantity are assessed using methods like UV absorbance and electrophoresis to ensure integrity for accurate sequencing outcomes.
The next step involves the creation of small RNA libraries. Small RNA molecules, including miRNAs, are enriched from the total RNA pool using size selection methods such as gel electrophoresis or size-exclusion chromatography. Adapters are then ligated to the enriched small RNA fraction, which facilitates reverse transcription into complementary DNA (cDNA) and subsequent amplification. These adapters often include unique barcodes or indexes, allowing for the multiplexed sequencing of multiple samples in a single run.
The prepared libraries undergo quality control to confirm the presence and concentration of miRNA libraries before sequencing. The sequencing process generates millions of short reads, typically 20-30 bases in length. Computational analysis of this data is then performed, which involves preprocessing and quality control to remove low-quality bases and potential contamination. This enables comprehensive profiling of known and novel miRNAs in a sample, accurate quantification of their expression levels, and the identification of miRNA variants or isoforms.
Revolutionizing Diagnostics and Research
Next-Generation Sequencing for microRNA analysis has impacted medical diagnostics and biological research. This technology has advanced the discovery of miRNA biomarkers for various diseases, including cancer, cardiovascular diseases, and neurological disorders. MiRNAs are attractive as biomarkers due to their stability in biological fluids and their tissue- or disease-specific expression patterns.
In oncology, NGS-based miRNA profiling provides insights across many cancer types, aiding in early detection, prognosis, and therapeutic monitoring. Distinct miRNA signatures can differentiate cancer types and stages, such as those found in glioblastoma or prostate cancer that correlate with progression.
Beyond cancer, miRNAs detected in biofluids are promising for diagnosing and monitoring neurological disorders. MiRNA expression profiles are also being investigated for their role in cardiovascular diseases. This understanding of miRNA expression patterns and their dysregulation holds potential for developing miRNA-based therapies, either by supplementing downregulated miRNAs with synthetic mimics or inhibiting overexpressed miRNAs with antagonists.