Ribonucleic acid, commonly known as RNA, serves as a molecular messenger within living cells. It carries instructions from deoxyribonucleic acid (DNA), which is the cell’s genetic blueprint, to guide the creation of proteins. These proteins perform most of the work in cells and are necessary for the structure, function, and regulation of the body’s tissues and organs. RNA provides a real-time snapshot of what a cell is doing at a particular moment, making it invaluable for understanding biological processes and disease states.
The Challenge of RNA Instability
RNA molecules are inherently fragile, making their preservation a challenge in scientific and medical fields. This instability largely stems from its unique chemical structure, specifically the presence of an extra hydroxyl group on the ribose sugar backbone. This 2′-hydroxyl group makes RNA susceptible to a process called autohydrolysis, where the molecule can spontaneously break down, especially in the presence of water or at higher temperatures. In contrast, DNA’s double-helix structure and lack of this extra hydroxyl group contribute to its greater stability.
Beyond its inherent chemical fragility, RNA faces a threat from ribonucleases, or RNases. These ubiquitous enzymes actively degrade RNA molecules, breaking them down into smaller components. RNases are resilient and found almost everywhere, including on human skin, in dust, and on most laboratory surfaces. Their widespread presence and activity even after harsh treatments like boiling or autoclaving make contamination a persistent concern. Trace amounts of RNases can rapidly compromise RNA samples, leading to a loss of valuable biological information.
Methods for Preserving RNA
Given RNA’s susceptibility to degradation, various methods have been developed to maintain its integrity for research and diagnostic purposes. One widely used approach is cryopreservation, which involves rapidly freezing samples to halt both enzymatic and chemical degradation. Flash-freezing in liquid nitrogen, which cools samples to approximately -196°C, or storing them in ultra-low temperature freezers at -80°C, effectively stops cellular activity and enzyme function. This extreme cold preserves RNA by slowing molecular motion to a near standstill, preventing its breakdown over extended periods.
Chemical stabilization offers an alternative to freezing, particularly for immediate sample handling and transport. Specialized reagents, such as RNAlater or similar aqueous solutions, permeate tissues and cells to inactivate RNases upon contact. These solutions contain high concentrations of ammonium sulfate and chelating agents like EDTA, which inhibit RNase activity and prevent spontaneous RNA hydrolysis. Samples treated with these reagents can be stored temporarily at room temperature for several days or at 4°C for longer periods, providing flexibility when immediate freezing is not possible.
Another method, used in pathology, is formalin-fixation and paraffin-embedding (FFPE). This process involves treating tissue samples with formalin, which cross-links proteins and preserves tissue architecture, followed by embedding them in paraffin wax. While FFPE is effective for morphological studies and long-term tissue storage, the chemical reactions involved can modify the RNA within the sample. This modification can make subsequent RNA extraction more challenging and affect the quality and integrity of the RNA, impacting downstream molecular analyses.
Proper Sample Handling and Collection
Preventing RNA degradation begins before long-term storage, with careful attention to sample handling and collection procedures. The primary principle is to create and maintain an environment free of RNases. This starts with speed, as RNA begins to degrade immediately after a sample is collected from its living source. Processing samples as quickly as possible, ideally within minutes of collection, minimizes the window for enzymatic activity.
Laboratories employ strict protocols to minimize RNase contamination. This includes using certified RNase-free lab equipment, such as sterile tubes, pipette tips, and collection vessels, which are treated or tested to be RNase-free. Researchers wear gloves and change them frequently, especially after touching skin or non-decontaminated surfaces, as RNases are present on human skin and hair. Work surfaces and instruments are regularly decontaminated using RNase-inactivating solutions or by baking at high temperatures to destroy enzymes. These proactive measures form a fundamental line of defense against RNA degradation.
Applications of Preserved RNA
The ability to preserve RNA integrity is essential for numerous applications in medical diagnostics and scientific research. In medical diagnostics, preserved RNA is routinely used to detect viral infections where the genetic material is RNA, such as influenza or the SARS-CoV-2 virus that causes COVID-19. The widely recognized Polymerase Chain Reaction (PCR) test for COVID-19 relies on stable RNA extracted from patient samples to identify the presence of the viral genome. Preserved RNA also plays a role in cancer diagnosis, where analyzing gene expression profiles from tumor samples helps clinicians understand tumor behavior, predict treatment responses, and monitor disease progression.
In scientific research, analyzing preserved RNA allows scientists to gain insights into gene expression in different cell types or under various conditions. Techniques like RNA sequencing provide a comprehensive view of the transcriptome. This information helps uncover the molecular mechanisms of diseases, identify targets for drug development, and further our understanding of biological processes. The integrity of RNA is necessary for accurate and reliable results in these complex analyses.