Streamlining the RNA Extraction and Purification Workflow

Any research project that ostensibly starts with a sample of purified RNA—such as RNA-seq and RT-PCR—inevitably begins earlier, with the process of obtaining the RNA from tissue or isolated cells. But extracting and purifying RNA from that important starting sample is no easy task. RNA is an unstable molecule that easily degrades unless care is taken to preserve it during the process of extraction. Further purification removes contaminants, DNA, and other unwanted molecules to obtain high-quality RNA samples needed for the best results in downstream applications. This article looks at optimized reagents, kits, and workstations that researchers rely on to streamline workflows for RNA extraction and purification.

Workflows

Extraction workflows—whether by the phenol/chloroform method, the spin column/chromatography method, or the magnetic beads method—begin with lysing cells or tissue material, and then separating the lysate into a fraction that contains RNA.

Cytiva offers workflows based on magnetic beads, adapted for a wide range of sample types, including tissue, eukaryotic/prokaryotic cells, liquid biopsy, and nasal swabs. Their workflows allow purification of specific types of RNA molecules, such as mRNA, microRNA, or viral RNA. Monika Seidel, Principal Investigator in Genomics at Cytiva, says that each sample type and analyte poses distinct challenges that are best addressed with a specific extraction process.

“Many difficult-to-lyse samples such as solid tissue or gram-positive bacteria require a pre-processing step that involves mechanical disintegration and/or enzymatic digestion to enable efficient release of RNA from the cells,” she says. “Similarly, the type of RNA to be isolated will dictate certain alterations of the extraction process; for example, fast and streamlined extraction of mRNA from eukaryotic cells can be achieved with oligo dT conjugated to the beads that specifically capture mRNA through its poly-A tail.”

NEB’s Monarch® RNA workflows are designed for the extraction and purification of RNA using varieties of sample types, columns, chemistries, and protocols. “Additionally, we have workflows for RNA cleanup that purify RNA from various reactions, including in vitro transcription, and labeling and capping reactions,” says James Deng, Senior Product Marketing Manager for NEB’s nucleic acid purification product lines. “These workflows are designed to clean up and concentrate RNA after enzymatic reactions, synthesis, and extraction protocols, including TRIzol.”

To isolate total RNA through purification, Bio-Rad Laboratories offers silica membrane-based Aurum kits (in mini column and 96-well plate formats), and the PureZOL™ RNA Isolation Reagent, which provides high-yield purified RNA. “For quick viral RNA extraction of biological samples for qPCR or digital PCR, molecular biology-grade Chelex 100 and Instagene are another great option,” says Anna Lai, Global Product Manager for PCR Reagents at Bio-Rad Laboratories. “Both have been used for many years in DNA extraction, and during the COVID-19 pandemic proved their utility for rapid, low-cost RNA extraction.”

Chroma’s MagXtract 3200 system has an open platform designed to put magnetic bead-based RNA (and DNA) extraction kits into action. It can automate all steps required for RNA extraction and purification, and downstream PCR setup. “MagXtract 3200 is an automated extraction system with liquid-handling capabilities that enable automatic distribution of primary sample to extraction kit, and distribution of product to storage tube or PCR plate to prepare PCR reactions,” says Elvy Fan, Senior Product Specialist at Chroma. “It greatly improves efficacy, reducing over 90% hands-on work, and delivering consistent and precise results.” Chroma has partnered with reagent manufacturers to validate extraction kits for various sample types, including virus, pathogen, blood, forensic, food, microbiome, plant, and stool.

Dealing with difficult samples

Unfortunately, sometimes the most precious samples (such as formalin-fixed, paraffin-embedded, or FFPE, tissues) yield RNA that is scarce, damaged, fragmented, or degraded. In these cases, pre-processing steps can boost extraction efficiency. Examples include removing paraffin from FFPE samples, disrupting complexes of proteins and nucleic acids, and digesting crosslinking proteins. “The fine balance between the removal of unwanted contaminants that might interfere with downstream applications, and RNA loss during excessive washing steps, often becomes a key determinant of success when dealing with limited samples,” says Seidel. “In cases of severely degraded samples, the prevalence of small RNA fragments calls for changes in extraction conditions and supplementation of lysis/binding buffers with additional compounds that promote the association between fragmented nucleic acids and binding matrices.”

Whenever possible, adhere to best practices for sample preparation, including cold temperatures and reagents to stabilize RNA. “Monarch DNA/RNA Protection Reagent is included in our kit and preserves RNA in solid and liquid samples for short and long-term storage, without the need to remove the reagent in the extraction workflow,” says Deng. “It’s also important to review the expected RNA yields, purity, and integrity from different tissue types and/or samples, as these could vary significantly, to better understand if the RNA extracted is in line with typical amounts for that sample type.”

Tools for researchers grappling with degraded RNA include amplification kits optimized for lower inputs of smaller RNA fragments. “For cDNA synthesis from RNA or pre-amplification prior to PCR, the iScript™ cDNA Synthesis Kit is a popular option with many customers,” says Lai. “This kit allows generation of cDNA with a combination of oligo(dT) and random hexamer primers that is suitable for samples with low RNA inputs and is optimized for fragments below 1 kb of length.”

To obtain more RNA from a degraded starting sample, Chroma’s MagXtract 3200 allows researchers to use a larger sample volume. Their patented method of mixing increases the potential processing volume by about 40%. The open platform affords flexibility to choose the best reagents and kits for particular degraded samples, and offers other features for executing varied protocols, including protocol editing. “The elution and lysis columns of its extraction decks can be heated up to 130℃, and the temperature can be set by users according to their selected extraction kits,” says Fan. “If necessary, MagXtract 3200 can automatically dispense internal controls or proteinase K, and reagents can be placed on the storage deck with cooling to 4℃ to maintain reagent integrity.”

Consider downstream applications

Your intended downstream application may influence your choice of extraction/purification method. For example, a transcriptomics researcher might use RT-PCR or next-generation sequencing (NGS) to analyze an mRNA sample. Total extracted RNA can be used in RT-PCR, but an additional purification step—namely, removal of ribosomal RNA (rRNA), which makes up around 80% of total RNA—is critical prior to NGS. This can be accomplished through rRNA depletion using specific oligos to capture and remove rRNA, or through mRNA positive selection employing oligo dT beads that will bind to poly-A tails. “Each method has its pros and cons,” notes Seidel. “However, the ultimate decision of which one to choose will be dictated by the initial sample type—for example, poly-A selection would not be suitable for a heavily degraded sample such as FFPE.”

Deng also recommends considering the volume requirements of your downstream plans. “If you require a more concentrated solution or a smaller volume, our workflows can be adjusted to elute RNA at lower volumes,” he says.

Degradation and preparation

Because high-quality RNA is of the utmost importance for successful RNA experiments, RNA degradation is your prime opponent. Any successful RNA protocol must contend with multiple sources of RNA degradation, so working in an RNase-free environment is a must. The chemical instability of RNA molecules at higher temperatures can be countered by cooling and temperature-controlled conditions. “The second and usually much more detrimental aspect of RNA instability in extraction workflows is the promiscuous nature of RNA degrading enzymes that are difficult to irreversibly inactivate,” says Seidel. “In this context the key to successful RNA isolation is inactivation of endogenous RNases that are released once the sample is lysed, and prevention of exogenous RNase contamination through the use of certified RNase-free consumables, PPE, and good laboratory practice.”

In addition, verify that your protocol works before embarking on extraction. “Check that your workflow can effectively lyse and extract RNA from your sample type, and ensure that your samples are completely disrupted and homogenized to release RNA and maximize yield,” says Deng. “Further, choose input amounts carefully based on recommendations for your sample type, as it is very important to not overload the column when extracting and purifying RNA— as yields, purity, and integrity may suffer.” This type of advance planning of your workflow will go a long way toward successful RNA experiments. “Beyond RNA extraction,” notes Lai, “the choice of reverse transcriptase and polymerase, as well as next-generation sequencing library prep kit, can influence the ability to obtain high-quality data even from limited or poor-quality RNA samples.”