What we will review in this blog:
Metatranscriptomics involves analyzing the transcriptome, which refers to all RNA molecules, produced by the microorganisms found in a specific environment, such as the gut, water, or soil. By using high-throughput RNA sequencing, researchers can gain a better understanding of gene expression patterns, metabolic activities, and the functional roles of various microbial species in the ecosystem. Although the main focus of metatranscriptomics is on messenger RNA analysis, it is possible to also analyze rRNA genes to target active communities within the samples.
The principles of metatranscriptomics are based on the assumption that RNA transcripts are a reflection of active gene expression. Researchers can determine the functional genes that are being expressed by microbes and learn more about the metabolic and cellular processes taking place in a microbial community by analysing the transcriptome. Metatranscriptomics also offers a dynamic perspective of microbial community functions by revealing variations in gene expression over time or in response to environmental stressors.
It is crucial to carefully choose the sampling location and time, where the activity of interest is anticipated to be taking place. This is particularly significant because messenger RNA is inherently unstable and has a limited lifespan. For instance, if the goal is to study cold resistance genes, sampling should occur after a transition to cold conditions. Moreover, to prevent mRNA degradation, it is necessary to stabilize the samples using either low temperatures or stabilization solutions.
Obtaining high-quality RNA for cDNA synthesis and sequencing heavily relies on the RNA extraction process, which is considered the most crucial step. RNA extraction is typically performed in a specialized laboratory with RNAse-treated instruments and controlled working environments. Following the extraction, rRNA depletion can be implemented to minimize the rRNA content and augment the mRNA content within the sample. Alternatively, RNA/DNA co-extraction can be utilized to facilitate paired metagenomics and metatranscriptomics sequencing.
Once total RNA has been extracted and purified, depleting mRNA is necessary to achieve sufficient sequencing coverage. There are two approaches to RNA library preparation: rRNA depletion or poly(A) mRNA selection. The latter option is favored as it conserves not only rRNA and mRNA but also snoRNA. Prior to sequencing, the RNA is fragmented, subjected to size selection, and converted to double-stranded cDNA via single-strand cDNA conversion and second-strand preparation. Due to RNA's delicate and transient nature, quality control (QC) checks must be performed after these processes.
The ultimate stage involves bioinformatics analysis. The manner in which metatranscriptomic data is processed and analyzed depends on the research questions being explored in a given study. In metatranscriptomics, bioinformatics pipelines are utilized to scrutinize the expression profiles of microbial communities in various environments.
Microbiome Insights has a team of experts available to help with your metatranscriptomic sequencing, from sample preparation to bioinformatics and data analysis. If you have any questions, get in touch with our team, who will be happy to help.