Ribonucleic acid, or RNA, is a molecule found in all living things. It plays numerous roles, from translating genetic instructions into proteins to regulating cellular processes. In a laboratory, an RNA sample is a purified collection of these molecules isolated from a biological source.
This extracted RNA provides a snapshot of cellular activity at a specific moment. Researchers use these samples to investigate the workings of cells in both healthy and diseased states. The quality of the sample is important, as it forms the basis for many scientific and medical investigations.
Sources of RNA Samples
Scientists obtain RNA from a wide array of biological materials, with the choice of source depending on the research question. For studies involving human health and disease, common sources include blood, tissue biopsies, and saliva. Blood is minimally invasive to collect, while tissue biopsies from tumors offer direct insight into a specific disease. Cultured cells grown in a laboratory provide a controlled and renewable source for experiments.
Beyond human and animal sources, RNA is also extracted from plants and microorganisms. Researchers might isolate RNA from plant leaves, roots, or seeds to study gene functions related to growth or stress responses. Microorganisms like bacteria and yeast offer insights into basic cellular processes and microbial genetics. The ability to extract RNA from viruses is used for diagnostics, as it allows for the detection of viral infections.
The RNA Extraction and Purification Process
The journey from a biological specimen to a usable RNA sample is a multi-step process designed to isolate the molecules while removing other cellular components. The first step is cell lysis, where cells are broken open to release their internal contents. This is achieved using detergents and mechanical force, creating a mixture of RNA, DNA, proteins, and other cellular debris.
Once the cellular contents are released, the purification phase begins to eliminate contaminants like DNA and proteins, which can interfere with analyses. One common method uses organic solvents like phenol and chloroform to separate the mixture into distinct layers, with RNA remaining in the aqueous phase. To ensure all DNA is removed, an enzyme called DNase is added to break down any remaining DNA, followed by several washes to remove residual salts and impurities.
The final step is elution, where the purified RNA is collected in a clean, stable solution and stored at very low temperatures. Because RNA is inherently unstable and susceptible to being broken down by enzymes called RNases, the process must be performed with care to ensure the final sample is of high quality.
Determining Sample Quality and Integrity
After extraction, it is necessary to verify that the RNA sample is suitable for experimental use. This quality control process assesses three main characteristics: quantity, purity, and integrity. A poor-quality sample can lead to inaccurate results, so this is a standard step in the research workflow.
The first aspect, quantity, measures the concentration of RNA in the sample. This is determined using spectrophotometry, where a device measures how much light is absorbed by the sample at a specific wavelength. This information tells researchers if they have enough material for their planned experiments.
Next, the purity of the sample is assessed to ensure it is free from contaminants left over from the extraction process. Spectrophotometry is also used here, with the ratio of absorbance at different wavelengths providing a reliable indicator of purity. Deviations from expected values signal the presence of contaminants that could interfere with experiments.
Finally, the integrity of the RNA is evaluated to determine if the molecules are intact or have been broken down. Degraded RNA can invalidate gene expression studies, making this a primary quality metric. The most common method for this assessment is the RNA Integrity Number (RIN), a score from 1 to 10 generated by an automated system. A higher number indicates a more intact, higher-quality sample.
Applications in Research and Medicine
Purified RNA samples are used in numerous scientific and medical advancements. One widespread application is in gene expression analysis. By measuring the amount of different types of RNA in a sample, scientists can determine which genes are active, or “turned on,” within a cell at a particular time. This provides a dynamic view of cellular function and helps in understanding diseases like cancer, where gene expression patterns are disrupted.
In diagnostics, RNA samples are used for tests that detect infectious diseases. The reverse transcription-polymerase chain reaction (RT-PCR) test used to diagnose viral infections like influenza and SARS-CoV-2 works by detecting the specific RNA sequences of the virus. The presence of this viral RNA in a patient’s sample confirms an active infection, allowing for rapid diagnosis.
RNA technology has also led to the development of new therapeutic strategies. The creation of mRNA vaccines is a prime example, where synthetic messenger RNA is produced to instruct the body’s cells to make a specific protein. This action triggers an immune response. These applications highlight how the study of RNA bridges basic research and tangible medical outcomes.