Guide to Common RNA Extraction Methods

Extracting RNA from a cell or tissue sample—the first step in RT-qPCR or RNA sequencing experiments—is detailed-oriented work warranting a dependable method. Though RNA extraction protocols abound, there are several that researchers come back to time and again. These varied and trusted methods provide different strengths depending upon a researchers’ needs—just the kind of diverse toolset one relies upon having in the lab’s toolbox. Here is a look at the basic steps of common RNA extraction methods, and expert perspectives on using these valuable tools.

Preparation and prevention

The moment tissues are homogenized and cells are lysed, RNases begin degrading RNA. Preserve your target RNA by optimizing lysis conditions, using RNase inhibitors, and taking sensible precautions to minimize RNase activity. “To limit RNase introduction, wear gloves when handling the reagents, supplies, and equipment for RNA extraction,” says Anagha Kadam, Applications and Product Development Scientist, Monarch Nucleic Acid Purification, New England Biolabs. “Also, use an RNase-inactivating reagent to wipe down the benchtop, pipettes, and equipment immediately before starting the RNA extraction.”

RNA extraction begins before the first step of the protocol. “Stored RNA is subject to degradation from RNases the moment it is released from the biological sample,” says Paraj Mandrekar, Technical Services Scientist at Promega. He advises using reagents to disrupt both the tissue and RNases, such as Promega’s simplyRNA Tissue Kit, which contains both Homogenization Buffer and 1-thioglycerol.

Remember to guard against RNases by cleaning work surfaces often. “We recommend at each step of handling the sample to apply detergents and RNase preventative measures to the lab bench and other surfaces, like RNase Zap Decontamination solution, and be ready to change your gloves frequently,” adds Mandrekar. “Only interact with tubes and plasticware wearing gloves and wash your gloved hands with detergent frequently.” Use an RNA stabilization solution, such as New England Biolabs’ Monarch StabiLyse DNA/RNA Buffer or Invitrogen RNAlater Stabilization Solution, and store at -80°C.

Common RNA extraction methods

During tissue homogenization and/or cell lysis (mechanically or with chaotropic agents), proteins are denatured and RNases are inactivated to preserve RNA. Then RNA is captured in an organic phase extraction, or by binding to a silica filter or magnetic beads. Non-RNA species are then removed, and the RNA is resuspended in buffer.

Here are some of the most common methods used today:

Guanidium acid-phenol extraction, a.k.a. organic phase extraction (e.g., Trizol reagent method). Though convenient RNA extraction kits are commercially available, this method is the most common one to be performed without a kit. This is a liquid-liquid extraction method that separates RNA to the aqueous phase and DNA/proteins to the organic phase.

Guanidinium thiocyanate is a chaotropic agent used to denature proteins. It’s one of the primary ingredients in Thermo Fisher Scientific’s Invitrogen™ TRIzol™ Reagent, which has been used for decades to isolate RNA, in addition to DNA and proteins. After homogenizing in TRIzol, the user adds chloroform and allows the solution to separate into phases. Other vendors sell similar reagents that work using phenol-chloroform extraction. This method offers the best RNA purity and yield, but the protocol tends to be more hands-on and lower throughput than spin column or magnetic bead procedures.

Silica filters and spin columns. This is a solid-phase extraction in which RNA binds to the silica in column-based spin filters (common in commercially available kits). Bound RNA can be washed and then eluted. These offer ease of use, moderate purity, and are amenable to both manual and automated processes.

Magnetic beads. Small (20–30 nm) magnetic beads coated in materials such as silica can bind RNA. A magnetic field holds the beads in place while RNA is washed and eluted. These work well for automation and high-throughput applications.

Hybrid systems. These combine Trizol-based lysis with spin columns or magnetic beads, offering the potential of higher RNA purity and yield with the ease of other systems.

Miodrag “Mickey” Micic, professor of engineering technology at Cerritos College, and chief technology officer at RotaPrep, notes that hybrid methods have led to advances in RNA extraction. “Combining organic phase separation—commonly referred to as Trizol methods—with spin column-based protocols allows for both high-quality and high-quantity RNA purification,” he says. “A similar approach has also been adapted for hybrid magnetic bead-based kits.”

To kit or not to kit

Most methods are commercially available in convenient kit form, though this isn’t strictly necessary. One advantage of kits is that they are optimized for RNA extraction in a particular situation or type of sample. “For example, Promega has several kits specific to isolating microRNA,” says Mandrekar. “In many cases, if we do not have a specific kit, we can make recommendations of how to approach the desired sample based on our understanding of the sample and research need, as well as our bank of application notes.”

Spin column kits are commonly used in smaller-throughput situations, such as smaller research labs and educational settings. Magnetic bead-based kits are often preferred in large facilities equipped for higher throughput and greater automation. Kadam recommends a kit that performs well with a wide range of sample types, such as New England Biolabs’ Monarch RNA kits. “Customers get an ‘all-in-one’ solution that works well with sample types including cell, tissues, microbes, plants, blood, and plasma,” she says. “Our kits include all the necessary reagents—including columns for gDNA removal, Proteinase K, and DNase I enzymes—which make for a simplified buying experience.”

Using kits for RNA extraction is increasingly common across all sample types. “Before kit-based extraction became the standard, organic extraction methods were used, which relied on phase separation and precipitation,” says Kadam. “But today, those methods are generally considered outdated since they are tedious, prone to variability, and generate more hazardous waste.” Initially it was believed that organic-based extraction methods worked better with some types of samples, such as lipid-rich tissues that are considered difficult to process. Today, however, kits such as the Monarch Spin RNA Isolation Kit has been formulated to extract various samples including lipid-rich tissues.

Because every experiment is different, there is no best RNA extraction method or kit. In general, Trizol-based extractions offer the best purity separation and yield, yet are labor-intensive and difficult to automate. But other factors are important too. “Spin column kits provide high yield, versatility, and simplicity, but offer lesser RNA purity than other methods, while magnetic bead-based systems excel in automation and high-throughput workflows, but offer lower yield and are [more] expensive,” says Micic. “Hybrid approaches and specialized kits continue to expand the possibilities for RNA extraction, addressing even the most challenging samples with innovative solutions.”

Compatibility with downstream applications

Most RNA extraction methods are likely to be compatible with most downstream applications, but Kadam advises verifying this. “For example, if you are interested in investigating small RNAs but also want to have the option of assaying for transcriptome changes, it is beneficial to use a kit that empirically supports the extraction of the total RNA pool, including <200 nucleotides," she says.

If you are extracting RNA for downstream RT-qPCR or RNA sequencing applications, it is important to use stringent wash steps to ensure high RNA purity. “This is particularly important if the downstream application is RT-qPCR, because any salts, proteins, or ethanol that are present in the RT-qPCR input can have a significant inhibitory effect on the reaction,” Kadam notes. Similarly, Mandrekar explains that the presence of phenol in phenol-based RNA extraction methods can affect some downstream processes. “This is one more reason to move away from older phenol-based isolation methods toward column- or particle-based approaches to RNA isolation,” he says.

However, that depends upon your experimental aims. Phenol-based or hybrid extraction methods also offer high-quality RNA extraction results that are important for applications such as next-generation sequencing, microarrays, and RNA structural studies, says Micic, while “spin column-based kits suffice for routine tasks like RT-qPCR, and magnetic bead-based methods excel in automation and high-throughput diagnostics.” With such guidelines in mind, it should be easier to pair the most appropriate RNA extraction method with the application for the best outcome.

Optimizing RNA extractions: Essential dos and don’ts

1. Prevent RNase activity. Use RNase inhibitors, clean surfaces with RNase-inactivating reagents, and always wear gloves to avoid RNA degradation.

2. Use RNA stabilization solutions. Store RNA samples in stabilization solutions like Monarch StabiLyse or Invitrogen RNAlater to prevent degradation.

3. Choose the right extraction method.

• Trizol (phenol-based): High purity but labor-intensive
• Spin columns: Easy to use, moderate purity
• Magnetic beads: Good for automation and high-throughput
• Hybrid systems: Combine Trizol with columns/beads for high yield and quality

4. Verify compatibility with applications. Make sure the extraction method is suitable for downstream applications like RT-qPCR or RNA sequencing.

5. Consider kits for convenience. Kits offer optimized solutions for specific samples and simplify the extraction process (e.g., Monarch RNA Kits).

6. Follow strict wash steps. To avoid contamination, ensure thorough washing to remove salts, proteins, and ethanol, especially for sensitive applications.