RNA isolation and purification

RNA isolation: Methods, challenges, and applications

RNA isolation is a critical process in molecular biology and biotechnology that involves the extraction of ribonucleic acid (RNA) from biological samples. RNA serves as a blueprint for protein synthesis and plays a vital role in gene expression and regulation. The purity and integrity of the isolated RNA are essential for downstream applications such as reverse transcription PCR (RT-PCR), RNA sequencing, and microarray analysis.

Understanding the nuances of RNA isolation is crucial because RNA is inherently unstable and prone to degradation. Successful RNA isolation requires careful handling, optimized protocols, and the right choice of reagents and equipment to overcome these challenges. Moreover, the diverse nature of biological samples, which ranges from microbial samples to preserved tissues, necessitates specialized considerations that fit well to each sample source or downstream application.


Mark: Hi Dr. Zhong, I was working on my RNA extraction experiments and noticed how tricky some samples can be. Could you help me understand the common challenges in RNA extraction and how to address them?

Dr. Zhong: Of course, Mark. RNA extraction can be challenging because RNA is highly unstable and easily degraded. Different sample types, like tissues, blood, or plants, add their own unique hurdles. Let’s go through these challenges and the strategies to overcome them. Come on, let’s begin!

RNA isolation typically involves several key steps:

  1. RNA stabilization: Involves steps that prevent RNA degradation, such as RNase inhibitors or stabilizing agents.
  2. Cell lysis: Also called ‘cell disruption,’ which involves breaking the cells open or disrupting the tissues to release RNA (e.g. use of bead mills). These steps are achieved through chemical, mechanical, or enzymatic methods, depending on the sample type and intended application.
  3. Separation: Includes processes that separate RNA from other biomolecules such as DNA, proteins, and other cellular components. A common method includes phenol-chloroform extraction, which isolates RNA into the aqueous phase, separating it from DNA and proteins.
  4. Purification: Involves removing contaminants such as salts (derivatives of several RNA extraction reagents) to obtain high-quality RNA. Common methods include additional wash steps with 70% ethanol or buffers to remove impurities when performing phase separation-based techniques.
  5. Elution or resuspension: Typically, the final step of the isolation process involves either eluting or resuspending the RNA to recover it in a usable form. Elution is normally carried out using a buffer with low salt concentration or nuclease-free water, allowing the RNA to be subsequently recovered and solubilized in a liquid solution. Meanwhile, resuspension typically begins with precipitating the RNA in the presence of salt, which also helps concentrate the extracted RNA. After washing and drying, the RNA is reconstituted by dissolving it in a suitable buffer (e.g., TE buffer or water).

In some workflows, such as the column-based method, RNA separation and purification can happen almost simultaneously in one procedure through subsequence binding, washing, and elution steps.

RNA isolation is fundamental to understanding cellular processes and disease mechanisms. It allows researchers to:

  • Analyze gene expression patterns, providing insights into how genes are regulated under different conditions.
  • Develop diagnostic tools for diseases like cancer, infectious diseases, and genetic disorders by detecting specific RNA markers.
  • Study the effects of treatments, environmental changes, or mutations on gene regulation, aiding in drug development and precision medicine.
  • Advance fields like transcriptomics, enabling comprehensive studies of RNA molecules in various cell types and tissues.
  • Facilitate the development of RNA-based therapeutics, including mRNA vaccines and gene-silencing approaches.

High-quality RNA is a prerequisite for reliable results in these applications, making RNA isolation a cornerstone of modern biotechnology and molecular biology research.

Several methods are used to isolate RNA, each with unique principles and suited to specific sample types and applications:

  • Phenol/chloroform extraction – an extraction method that uses organic solvents to separate RNA based on the differential solubility of cellular components. Note: Phenol-based methods have historically been widely used for RNA isolation, but they are now
    considered less favorable due to their toxicity to humans and the environment. Safer and more efficient alternatives are available today for RNA isolation.
  • Column-based method – involves using silica membranes or filters in a centrifuge to preferentially bind and elute RNA.
  • Magnetic bead-based method – employs magnetic particles coated with RNA-binding surfaces to capture RNA easily from the solution.
  • Automated RNA isolation – machines such as EZ2 Connect automate the binding, washing, and elution steps, ensuring consistency and scalability in the RNA extraction process.
Selecting the right method for RNA isolation is essential for achieving high-quality results in downstream applications. Each approach, whether using phenol-based extraction, RNA purification kits, magnetic beads, or automated systems, offers distinct advantages based on efficiency, hands-on time, cost, safety, and sample throughput. The table below presents some of the comparisons of different RNA isolation methods.
Key features of the different RNA isolation methods.
Method
Efficiency
Hands-on time
Cost
Safety
Sample throughput
Phenol-based extractionHigh yield***HighLow (basic reagents)High risk (toxic solvent)Large sample volumes
Spin column kitsHigh purityMediumModerate (requires proprietary RNA purification kits)Low riskSmall to medium
Magnetic beadsHigh target specificityLowHigh (initial setup) **Low riskMedium to high*
Automated systemsHigh reproducibilityMinimalHigh (initial setup) **Minimal risk (fully enclosed systems)Small to high*
* Indicates scalability for throughput.
** Indicates a low per-sample cost (on average).
*** Indicates that the RNA may contain carry-over salts, requiring further RNA purification.
Extracting RNA from various sources requires understanding the challenges and complexity of the RNA sources to address the challenges of extraction procedure. Below is a summary of challenges associated with each source and strategies to overcome them:
Sample Source
Challenges
Considerations
Human and animal tissuesRNase activity, variability in tissue composition, and rapid RNA degradationProcess samples quickly and store tissues in RNAprotect or flash-freeze them
Blood and bodily fluidsHigh protein content, cellular diversity, and presence of RNasesEmploy specialized kits and use recommended buffers
Plant samplesTough cell walls and high levels of secondary metabolites (e.g., polyphenols)Use bead-beating technique
Microbial samplesVariability in cell wall structures and compositions (Gram-positive or negative bacteria, fungi)Tailor lysis methods (mechanical, enzymatic) to the organism type, as previously described
FFPERNA fragmentation and cross-linking due to fixationOptimize deparaffinization and use RNA purification kits designed for FFPE RNA recovery such as RNeasy FFPE Kit.
Environmental samplesPresence of inhibitors (e.g., humic acids, organic matter)Use RNA purification kits with inhibitor removal steps specific to soil, water, or other environmental samples.
Cell culturesRNase contamination and potential for low RNA yieldProcess cells immediately or store in stabilizing solutions, ensure thorough lysis.
RNA extraction can be challenging, as different applications and sample types often have unique obstacles. Issues like genomic DNA contamination, low RNA yield, degraded RNA, or missing target RNAs can significantly impact downstream results, such as RT-PCR, RNA sequencing, or gene expression analysis. Identifying the specific challenges in your workflow and adjusting protocols accordingly is essential to optimizing your RNA isolation process.
Recommendations in overcoming common challenges in RNA isolation.
Problem
Recommendation
Tweaks and tricks
Purity (gDNA contamination, salt carry-overs, and inhibitors)Use spin column kits with gDNA removal steps to minimize contamination.Include a DNase treatment step during or after the extraction or use a kit with gDNA Eliminator Spin Columns
Use high-purity reagents and nuclease-free consumables. Use specialized kits, such as the RNeasy PowerClean Pro CleanUp Kit or magnetic bead-based methods, to efficiently eliminate a wide range of inhibitors.
Concentration (low yield)For low yield due to inefficient sample lysis and RNA recovery, switch to a method optimized for the sample type. For low yield due to a small sample volume, choose the right RNA Micro kits designed to purify RNA from very small samples.Increase input sample volume or optimize lysis conditions.
Integrity (degraded samples)Minimize handling time and avoid freeze-thaw cycles.Use fresh or properly stored samples.
Include a sample stabilization step before the extractionWork quickly and keep samples on ice throughout the protocol.
Target representation (missing target RNAs)Use specific methods that are designed to isolate the target RNA species, such as the use of miRNeasy Kit for miRNA Purification when working with miRNA or the use of OligodT magnetic bead-based kits when isolating mRNA.Combine two or more methods of RNA extraction when necessary to optimize the extraction of target RNA species.