A purified sample of RNA is an important starting point for many widely used assays today, from RT-qPCR to next-generation sequencing. While there are only three main approaches for extracting and purifying RNA from cellular samples, there are innumerable considerations that can help you optimize your workflow when isolating RNA. In this article, some experts weigh in with tips for purifying high-quality RNA to boost your confidence in subsequent assay results.

Main approaches to RNA extraction and purification

While the choice of extraction method can sometimes depend upon the sample type, generally these three approaches can be used with most samples. Even cell types that are more difficult to lyse and extract RNA, such as bacteria, plants, and yeast, can be successfully extracted using additional lysis steps.

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The phenol/chloroform method

This is the oldest, tried-and-true method for extracting RNA. It uses a straightforward yet laborious protocol that relies on the differing solubilities of molecular species in organic solvents and water. The phenol/chloroform method can accommodate larger samples, but it is low throughout and not easily automated. Incubation times and precipitation temperatures are important, says Matteo Beretta, molecular biologist at PCR Biosystems, who uses low temperatures to protect the RNA from degradation by RNAses. “Attention should be paid not to disturb the phases formed during the process [to prevent contamination of RNA with DNA or proteins], so a good handling ability is required,” says Beretta. “The phenol/chloroform method usually yields a slightly cleaner RNA and allows the extraction of RNA from a lower amount of cells [compared to the spin column method below].”

The spin column/column chromatography method

The spin column method uses small centrifuge tubes that contain silica-based filter columns. Lysed samples treated with RNase inhibitors and high salt concentrations are passed through the spin columns during centrifugation, while the RNA remains bound to the silica within the column. Washing removes impurities, then the purified RNA is eluted from the silica filter using water.

Spin columns, widely available in a convenient kit format, typically use a simple, quick protocol. But this can be complicated by incomplete sample lysis or homogenization, or overloaded filters. “It is important to use an appropriate amount of input material since using too much sample may reduce lysis efficiency, introduce excessive amounts of cellular components other than RNA, and compromise RNA binding to the RNA purification column,” says Danielle Freedman, senior product marketing manager at NEB. Automation is possible, but it is not as convenient as automating the magnetic beads method (see below).

The spin column method is only suitable for small samples, but larger amounts of RNA (e.g. grams to kilograms) can be purified by column chromatography using various resins. For column chromatography based methods, says Kelly Flook, senior product manager at Thermo Fisher Scientific, “the most important step in the purification workflow is optimizing load and elution steps for the best capacity and yield while maintaining high purity.”

Optimizing salt concentrations is particularly important when purifying mRNA by column chromatography. At higher salt concentrations, mRNA can precipitate or form higher-ordered structures like dsRNA. “When developing loading protocols for oligo(dT) affinity [columns,] it is helpful to first determine the salt concentration at which the mRNA precipitates or forms double strands, [as] loading below this concentration aids recovery and purity,” says Flook. “The level of salt required to bind to oligo(dT) is related to the number of nucleotides that need to be neutralized.”

The magnetic beads method

This method uses magnetic beads coated with silica or other ligands that bind RNA, such as oligo(dT) to isolate mRNA molecules with intact polyA tails. The beads are incubated with cell lysate and RNase inhibitors, then anchored in place using a magnetic field while the supernatant (containing unwanted debris and impurities) is removed, and the beads are washed to remove lingering impurities. Resuspending the beads in water elutes the purified RNA.

This is a quick, simple protocol that is easy to automate for high-throughput work. The lack of a filter column obviates concerns over filter clogging or overloading. More viscous samples can pose a challenge because it may be more difficult for beads and RNA to make contact during incubation.

Common pitfalls and concerns

Inhibiting RNases

The first concern in any RNA workflow is to guard against degradation by RNases. “Work in an environment which is as RNase-free as possible, so wash surfaces and pipettes, use RNase-free (DEPC-treated) water, and change gloves a lot,” says Beretta. Also, add the power of RNase inhibitors as needed. “RNase control is key, so the use of inhibitors specifically, or knowing which conditions cause inactivation of RNases, such as lysis buffers or transport media, together with use of RNase-free consumables, helps maintain [RNA] integrity,” says Andrew Gane, genomics and diagnostics solutions strategy and technology manager at Cytiva.

Sample lysis

The sample type will dictate the appropriate lysis stringency, which can vary widely. This may require optimization, as insufficient lysis means an incomplete yield, while overly stringent lysis can degrade RNA molecules. “The lysis efficiency can be fine-tuned by combining chemical lysis with enzymatic lysis and physical lysis via heat and/or mechanical disruption,” says Markus Sprenger-Haussels, VP, head of sample technologies product development life sciences at QIAGEN. “These parameters have to be well balanced to avoid negative impact on RNA integrity.”

Elution

Elution conditions should be optimized to find the best elution buffer for long-term RNA stability, and also to avoid interference with subsequent downstream applications. For example, azide can affect quantification by spectrophotometry, EDTA can impact PCR efficiency, pH can affect enzymatic reactions, and “addition of carrier RNA might impact [spectrophotometric] quantification or oligo(dT)-primed downstream reactions,” says Sprenger-Haussels.

Contamination with gDNA

Removal of residual genomic DNA (gDNA) from RNA preparations is also an important consideration for some downstream applications, and optimizing workflows can help to reduce gDNA contamination. “Genomic DNA may be carried over from the interphase of organic extractions, or when solid-phase RNA purification methods are overloaded,” says Freedman. “To remove traces of genomic DNA from RNA preparations, samples should be treated with DNase I.”

Use multiple optimization parameters

Sprenger-Haussels recommends evaluating multiple parameters when optimizing, not solely maximum yield quantified by spectrophotometry. Other parameters to consider include RNA degradation, and co-purification of small RNA or genomic DNA, evaluated by capillary gel electrophoresis; inhibitor carry over using PCR quantification; and genomic DNA carry over quantified using PCR and RT-PCR. “Only if you look at all of these parameters will you find the best possible process parameters,” says Sprenger-Haussels. “If you optimize a procedure only based on maximum yield, you will most likely end up with a process delivering degraded RNA, since degraded RNA has higher UV adsorption than intact larger RNA.”

The multiple parameters and tools available today make RNA extraction and purification more accessible and customizable than ever, giving every researcher the chance to obtain the highest-quality RNA possible.