RNA molecules are fundamental components within all living cells, playing diverse roles in carrying genetic information and facilitating cellular processes. Among the various types of RNA, ribosomal RNA (rRNA) holds a prominent position due to its abundance and direct involvement in a central biological function. While rRNA is important for life, its presence can sometimes hinder scientific investigations aimed at understanding other, less common RNA molecules. For this reason, the selective removal of rRNA has become a standard practice in many molecular biology experiments and analyses.
What is Ribosomal RNA?
Ribosomal RNA is a type of RNA molecule that forms the structural and catalytic core of ribosomes, the cellular machinery responsible for protein synthesis. These molecular factories are found in the cytoplasm of all cells, from bacteria to humans, where they translate genetic instructions from messenger RNA (mRNA) into proteins. Specific rRNA molecules, such as the 16S and 23S rRNAs in prokaryotes or the 18S, 5.8S, 5S, and 28S rRNAs in eukaryotes, assemble with various ribosomal proteins to create the functional ribosome.
Ribosomal RNA is distinct from other RNA types due to its abundance within a cell. It constitutes 80% to 90% of the total cellular RNA content. This high concentration, coupled with its stable structure, makes rRNA a dominant component in any total RNA extraction, posing a challenge when studying rarer RNA species.
Why Remove Ribosomal RNA?
The overwhelming abundance of ribosomal RNA poses a significant hurdle in molecular biology experiments, particularly those focused on analyzing other RNA types like messenger RNA (mRNA) or various non-coding RNAs. When a total RNA sample is used for analysis, the vast majority of the data collected will originate from rRNA sequences. This high proportion of rRNA can obscure the signals from less abundant but more informative RNA molecules.
For instance, in gene expression studies, researchers are primarily interested in quantifying mRNA levels, as mRNA carries the genetic code for proteins. If rRNA is not removed, it can consume a disproportionate number of sequencing reads or detection signals, reducing the effective depth of coverage for mRNA and other target RNAs. This leads to inefficient use of resources and makes it difficult to accurately measure the expression levels of thousands of genes. Removing rRNA improves the signal-to-noise ratio, allowing for a clearer and more precise examination of the RNA molecules of interest.
How Ribosomal RNA is Removed
Removing ribosomal RNA from a total RNA sample employs methods based on sequence-specific binding or enzymatic degradation. One widely used approach involves hybridization-based methods, which leverage the unique sequences of rRNA molecules. In these techniques, specially designed probes (DNA or RNA oligonucleotides) are synthesized to be complementary to known rRNA sequences. These probes are then introduced to the total RNA sample, where they bind specifically to the rRNA molecules through base pairing.
Once probes bind to rRNA, the rRNA-probe complexes can be selectively removed. A common strategy involves attaching probes to magnetic beads, allowing physical separation of the rRNA complexes using a magnetic field. Another method uses enzymes like RNase H, which specifically degrades the RNA strand within an RNA-DNA hybrid, destroying the rRNA once it has bound to a DNA probe. These hybridization methods are specific, targeting and eliminating only rRNA while leaving other RNA species intact. The goal of these strategies is to enrich the sample for non-rRNA molecules, such as messenger RNA and various non-coding RNAs, enabling more focused downstream analysis.
Uses of Ribosomal RNA Removal
Ribosomal RNA removal is a key step in numerous molecular biology techniques, allowing scientists to gain deeper insights into cellular functions and disease mechanisms. One prominent application is in RNA sequencing (RNA-seq), a method used to quantify gene expression across entire transcriptomes. By depleting abundant rRNA, RNA-seq libraries are enriched for messenger RNA, ensuring most sequencing reads correspond to protein-coding genes. This enables precise and comprehensive measurement of gene activity, essential for understanding biological processes and identifying disease biomarkers.
Beyond mRNA analysis, rRNA removal is also important for studying non-coding RNAs (ncRNAs), a diverse group of RNA molecules that do not code for proteins but play regulatory roles. Since many ncRNAs are present at much lower concentrations than mRNA, their detection and analysis would be hampered by the overwhelming presence of rRNA. In metatranscriptomics, which analyzes active genes within microbial communities, rRNA removal is important for removing host-derived rRNA from environmental or clinical samples. This allows researchers to focus specifically on microbial RNA, providing a clearer picture of microbial activity and interactions within complex ecosystems.