Isolating RNA from Difficult Samples

Isolating high-quality RNA is a meticulous task that is further complicated when working with problematic samples and tissues. Each tissue type contains an array of biological materials introducing unique challenges to RNA isolation. This often necessitates extra manipulation, precise handling, and incorporation of specific reagents during the extraction procedure. In this article, we address common challenges and suggest troubleshooting strategies to aid in the successful isolation of RNA from these difficult samples.

Challenging samples

Clinical and animal specimens

The complexity of RNA isolation is significantly heightened when dealing with a diverse range of clinical and animal samples. Brad Hook, Manager of Global Scientific Applications at Promega, identified a variety of these troublesome sample types, such as fibrous tissues or fatty tissues, which are problematic because they are hard to break down and lyse. Moreover, low biomass samples, such as fine needle aspirates or small organism tissues, offer a minimal amount of RNA due to their lower input. Large-volume samples, like those isolating cfRNA from urine, pose a similar problem; the extraction is laborious and may result in a low concentration of RNA.

Hook also identified one of the most troublesome sample types—formalin-fixed, paraffin-embedded (FFPE) tissues. Expanding on this, Anna C. Lai, Global Product Manager of PCR Reagents at Bio-Rad Laboratories, stated how FFPE tissues, frequently used in clinical workflows, are difficult to work with due to degradation and crosslinking from storage and processing. The formaldehyde in formalin can cause nucleotide modification and fragmentation. A number of factors such as fixative pH, tissue fixation duration, tissue block storage conditions, and extraction methods impact the quality and integrity of RNA.

Lai also detailed the complications encountered with cellular materials or biofluids. Specifically, fat-rich tissues (found in the brain, breast, and adipose tissues), fiber-rich tissues (such as skin, heart, and skeletal muscle), and RNase-rich samples (like blood, pancreas, and fungi) present considerable hurdles in the isolation process and can lead to lower yields of RNA.

Samples from applied science and environmental studies

Katherine Mechling, Molecular Workflow Tools Technology Development Scientist at MilliporeSigma, discussed the challenges of RNA isolation from some environmental sample types. She explained how the process of extracting RNA can be further complicated by the sample matrices. For instance, plant tissues require physical homogenization to break down cell walls, and the naturally released phenolic compounds during RNA isolation can degrade RNA or inhibit analysis.

Hook also detailed how these phenolics from plants and other compounds extracted from soil can lead to amplification and downstream assay inhibitors. Other common challenges include insects that are difficult to lyse due to their exoskeletons and sample types with very large volumes like viral RNA from wastewater.

To address the complex purification process of RNA isolation, Lai noted several key considerations. The first is to ensure comprehensive cellular disruption, which efficiently eliminates proteins, fat, and genomic DNA. Next, prevent phenol or alcohol carryover into the isolated RNA, and take care to successfully neutralize RNAses. Lai emphasized that all attributes of the purification process require evaluation toward enabling easy and efficient isolation of high-quality total RNA.

Techniques for successful isolations

While each sample and tissue type presents its own unique set of challenges associated with RNA isolation, there are a number of practical strategies that researchers can employ to ensure success. For tough-to-lyse samples, Hook recommended using liquid nitrogen. “Grinding works well to not only lyse the host sample but also prevent RNA degradation due to heat. The downfall is that this method is very low throughput and not everyone has convenient access to liquid nitrogen.” As an alternative, he explained that bead beating offers higher throughput, but requires optimization and can cause sample shearing or degradation due to heat and the physical nature of bead beating.

Conversely, heat can be used to improve the isolation in some samples. For tissue types such as lung and adipose, Hook suggested that applying heat in the presence of a lysis buffer could lead to improved outcomes, due to the stabilizing elements present in the solution.

Another popular method includes RNA isolation by using a monophasic solution of phenol and guanidine isothiocyanate (GITC), Lai explained. Purifications using spin-based silica membrane technology are also well-established and easy-to-perform means of isolation. However, she noted that there are drawbacks to both technologies. While GITC quickly and effectively lyses even difficult cellular membranes, RNA is often co-precipitated with contaminants that could compromise downstream applications. Spin-based silica technology delivers higher purity isolation, but drawbacks can include limitations on sample load and more importantly, inadequate disruption of homogenization of sample.

In particular, Lai advocated for using an extraction kit with a hybrid protocol like the Aurum Total RNA Fatty and Fibrous Tissue Kit for these samples, as they combine the benefits of an organic extraction and silica membrane technology to yield pure, contaminant-free RNA.

Mechling supported using various RNA extraction kits, tailored to specific sample types to enhance analysis accuracy. She specifically mentioned the utility of specialty plant kits and tissue kits. While the former removes contaminants like phenolic compounds and polysaccharides, the latter aids in the release of RNA and prevents analysis inhibitors. However, if using a phenol-chloroform extraction method like TRI Reagent^®, she advised cleaning up any residual phenol with a spin column like GenElute™-E Single Spin RNA Cleanup Kit.

Similar filter-based isolations and specific sample-type kits can help deal with low-input and high-volume samples. For low-input samples, using carrier RNA may also improve RNA recovery. When dealing with FFPE tissues, commercially available kits specifically designed for such samples should be utilized, such as the ReliaPrep™ FFPE Total RNA Miniprep System. These kits often contain reagents that reverse formaldehyde modifications. Special care must also be taken to avoid overheating or repeated freeze-thaw cycles of the tissues. Finally, to combat amplification inhibition, Hook recommended purification chemistries and amplification master mixes designed to reduce the effect of inhibitors in RNA eluates.

Additional considerations

Although all previously discussed tips and techniques assist with specific sample types, there are a number of recommendations for extracting high-quality RNA regardless of its origin. One crucial aspect is sample storage. “The most important factor is that poorly treated samples will result in low-quality RNA,” Hook emphasized. “This is especially true with fresh, frozen tissues.” He explained that tissues not stored in protective reagents like RNAlater, or those that have gone through repeated freeze-thaw cycles will produce lower-quality RNA. Furthermore, using RNasin in the isolated RNA, he noted, can help protect it in downstream assays.

Mechling suggested creating an “RNA only” workspace and further protecting the RNA by decontaminating the area against RNAases. “Researchers should also be sure to use RNase-free reagents to minimize introducing RNases into the prep, and include RNase-free DNase or other genomic DNA removal step during purification to avoid any genomic DNA contamination,” she noted.

Lai echoed the need for an RNase-free workspace and warns of the hazards of processing RNA samples that still retain gDNA. “PCR cannot distinguish between cDNA and contaminating gDNA of the same sequence, resulting in a false positive or overestimation of RNA present if primers are not designed properly.” Therefore, she recommended using an isolation kit with an integrated DNase I for gDNA removal, which also avoids exposing the RNA to high temperatures.

“Begin with your end application in mind and plan your experiments so you can work quickly,” Mechling advised. “Choose a kit or purification method that provides complete lysis and then add in a clean-up procedure to ensure minimal contaminant carryover.”

Table. Solutions for Addressing Difficulties with RNA Isolation