Best practices for RNA storage and sample handling

While the single-stranded nature of RNA provides it with the flexibility to perform a wide range of biological functions, it also makes it susceptible to degradation. RNA is vulnerable to RNases (enzymes that are both ubiquitous in biological samples and highly stable in various conditions) and hydrolysis caused by chemical or environmental factors such as high temperature or humidity. Thus, working with RNA requires careful planning and rigorous adherence to protocols. Following these best practices will help ensure high-quality yield in your RNA isolation protocol:

1. Designate a dedicated workspace: Use a clean, RNase-free area specifically for RNA work. Avoid general-use benches to minimize contamination risks.
2. Keep work surfaces and tools clean and sterilized: Regularly clean workbenches with RNase-deactivating reagents like disinfectant and alcohol. Ensure pipettes, electrophoresis tanks, and other reusable tools are thoroughly cleaned and treated to eliminate RNase residues.
3. Use disposable equipment: To reduce contamination risks, opt for single-use, RNase-free consumables such as plastic tubes and pipette tips whenever possible.
4. Protect samples from environmental exposure: Work quickly and efficiently to limit RNA exposure to the environment. Always keep tubes closed and avoid extended exposure to air. Ensure that all tubes containing RNA are kept on ice. Additionally, consider using RNA stabilization reagents or methods to preserve the integrity of RNA during sample collection, transport, and storage.
5. Use appropriate protective measures: Always wear disposable gloves, replace them frequently, and consider additional protective equipment if required for specific procedures.
6. Use nuclease-free, ultra-filtered, or autoclaved solutions and reagents: Ensure all buffers, water, and reagents used in RNA work are certified RNase-free, filtered, or autoclaved to eliminate any potential RNase contamination.

Ribonucleases (RNases) are active enzymes with specificity for RNA molecules that generally do not require cofactors to function. It is recommended to use plasticware and glassware that are RNase-free or had RNase deactivated first. For some protocols, such as the RNeasy Mini Kit, standard tubes can be used as the buffer components of the kit, such as guanidine isothiocyanate, can effectively inactive RNases. Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure. To create and maintain an RNase-free environment, the following steps can be implemented:

1. Personal hygiene: Always wear disposable gloves and replace them regularly, especially after touching non-sterile surfaces. Avoid breathing or speaking directly over open samples.
2. Equipment preparation: Treat non-disposable plasticware with 0.1 M NaOH/1 mM EDTA, followed by rinsing with RNase-free water. Autoclave glassware and treat with DEPC, if applicable. Rinse thoroughly to remove residual DEPC before use.
3. Clean workspaces: Before and after experiments, use RNase-deactivating reagents to clean all surfaces, pipettes, and tools. Wipe down workspaces regularly to prevent the accumulation of contaminants.
4. Sterile reagents: Only use reagents certified as RNase-free. Prepare aliquots to prevent repeated exposure to contaminants.
5. Pipetting techniques: Avoid reusing pipette tips, even if they are autoclaved, as they may carry RNase residues. Use fresh, disposable tips for each operation.

Sample handling is a critical step that can significantly influence the quality of RNA yield and the accuracy of downstream applications. Mishandling can lead to RNA degradation or inaccurate gene expression profiles. When handling samples for RNA isolation, keep the following guidelines in mind.

1. Sample stabilization: RNA is highly susceptible to degradation immediately after sample collection due to RNase activity and environmental factors. To preserve RNA integrity, use stabilizing agents or flash-freeze samples in liquid nitrogen. Ensure the use of RNase-free containers and maintain samples on ice or until stabilization is achieved. It’s important to note that most RNases are endogenous, originating from within the sample itself, making sample stabilization a crucial step in preserving RNA integrity during extraction.
2. Minimize processing time: RNA degradation begins immediately after the sample is harvested. Rapidly process biological samples to stabilize RNA as quickly as possible.
3. Use appropriate disruption methods: Employ appropriate methods for disrupting tissue samples, such as grinding in liquid nitrogen or using bead mills. Sample sources for RNA extraction are diverse, and homogenization efficiency depends on the specific sample type. Plant tissues, for example, require specialized methods for cell disruption due to their rigid cell walls and the presence of secondary metabolites, which contrast with the simpler homogenization procedures typically used for animal or human cells, which have softer cellular structures and fewer inhibitory components. Follow the recommended acclimatization and working temperature of the buffers and equipment to prevent heat-induced degradation. When working with high molecular weight RNA, avoid applying excessive mechanical force (e.g., passing of the cell lysate in a syringe and needle) to prevent shearing the RNA.
4. Avoid freeze-thaw cycles: Handle frozen samples under frozen conditions until lysis or stabilization occurs. Thawed samples can lead to RNase activation or heat-induced hydrolysis, resulting in RNA degradation.
5. Prevent cross-contamination: Clean homogenizers and other tools between samples to eliminate carryover. Use lysis buffers containing RNase inhibitors to protect RNA integrity during processing.

The presence of a 2’-OH (hydroxyl) group in the ribose moiety of the RNA molecule makes it susceptible to hydrolysis when subjected to heat treatment. High temperature causes the hydroxyl group to react with the phosphate, breaking the bonds between nucleotides and causing RNA degradation (3). Therefore, proper storage of RNA and RNA-containing samples is essential for preserving their quality over time.

It is crucial to flash-freeze biological samples immediately after collection using liquid nitrogen or add stabilization reagents to halt enzymatic activity. If the samples are processed immediately, keep them on ice until use. For short-term storage, keep samples frozen or use stabilization reagents, which can maintain RNA integrity at room temperature for limited periods. For long-term preservation, storing and freezing samples in a stabilization reagent is highly recommended. This method ensures excellent preservation and provides additional protection to RNA during the thawing process.

For purified RNA, divide it into small aliquots to avoid repeated freeze-thaw cycles, which can lead to RNA degradation. Store these aliquots in RNase-free water or TE buffer at –20°C for up to a few weeks or at –70°C for long-term storage. Additionally, ensure that storage containers are tightly sealed to prevent moisture buildup or contamination, and regularly monitor freezer temperatures to maintain consistent conditions.

RNA stabilization is a critical step for ensuring that the integrity and amounts of RNA in the sample are preserved throughout the extraction process. This is very crucial in studies where gene expression profiling is the main objective. Improper stabilization can result in false experimental outcomes due to degradation or induction artifacts.

One major concern for stabilizing RNA in the sample is the specific non-enzymatic hydrolysis by divalent cations (4). Mg2+ is one of the most abundant divalent cations in cells that catalyze the in-line attack of oxygen in the 2’-OH group on the phosphate backbone of RNA (5). It is then very crucial to use buffers that are free from divalent cations. In cases where these cations are present, the samples can be stabilized by the addition of chelating agents such as EDTA. Furthermore, RNAprotect can be used to stabilize RNA immediately after sample collection, preventing degradation and allowing for subsequent processing at a convenient time.

However, it is also important to note that different sample types require various stabilization reagents or methods to efficiently preserve RNA integrity during sample collection and/or sample storage. For instance, PAXgene tubes are a common choice for blood samples. On the other hand, tissue samples require stabilization reagents like RNAprotect. Several sample types (e.g., blood, animal cells, or plant tissues) present unique challenges for effective RNA isolation. Therefore, selecting the appropriate stabilization reagent is essential to obtaining high-quality RNA for downstream applications. It is also important to note that not all stabilization and RNA isolation methods are compatible with each other. In some cases, the components of the stabilization reagent may interfere with RNA isolation procedures. It is then crucial to choose compatible methods to ensure optimal RNA recovery and quality.