Best Practices for Efficient RNA Extractions

Key Takeaways

1. Efficient RNA extraction is crucial for reliable results in molecular biology workflows, but faces challenges such as degradation, contamination, and low yields.

2. Common issues in RNA extraction include RNA degradation (indicated by low RIN), contamination from proteins or DNA, and insufficient yields for downstream applications.

3. To address these challenges, researchers should ensure high-quality starting material, create an RNase-free environment, and choose gentle isolation methods that maximize yield while removing inhibitors.

4. Different sample types (e.g., blood, plant material, FFPE samples) require specific extraction techniques and considerations due to their unique compositions and structures.

5. When selecting an RNA extraction method, researchers should consider factors such as downstream experiment requirements, speed, sustainability, and overall cost.

6. Recent innovations in RNA extraction include streamlined protocols, advancements in extracting RNA from challenging sample types, and methods developed in response to the SARS-CoV-2 pandemic.

Efficient RNA extraction is an essential first step in numerous molecular biology workflows, ensuring pure and intact RNA for reliable results in downstream applications such as qPCR, RNA sequencing, and microarray analysis. However, this process can encounter several challenges, including degradation, contamination, and low yields. By employing best practices, proper techniques, and high-quality extraction kits, researchers can overcome these obstacles and ensure the accuracy and reproducibility of their results.

Frequent challenges and solutions

Researchers face numerous hurdles when extracting RNA. “One of the most common challenges is RNA degradation, indicated by a low RNA integrity number (RIN),” stated Nike Bahlmann, Director of Global Support & Sales U.S. at BioEcho Life Sciences. The instability of RNA, mainly due to its chemical structure and the abundance of RNases, makes degradation a common concern. In addition, Bahlmann noted that contamination from proteins or DNA could negatively affect any downstream experiments. This type of contamination can lead to enzyme inhibition, reduced sensitivity, and misinterpretation of results due to false positives in RNA-based assays. Another common pitfall in RNA extractions is low yields. Many downstream applications require a minimum amount of RNA, and studies involving rare transcripts may suffer due to low yields.

Fortunately, Bahlmann believes that there are three main ways these challenges can be addressed. The first is to ensure the starting material is of high quality. “If garbage goes in, there is no chance that garbage will not come out,” Bahlmann stated. For assessing integrity, Bahlmann recommends checking either the RIN or DV200 value, which provides insights into fragment size and suitability for sequencing. Researchers should look for clear and sharp ribosomal bands, particularly with FFPE samples, and high DV200 values to indicate sufficient quality. Additionally, spectrophotometry and fluorometric analysis can be employed to identify contamination from proteins or DNA.

Secondly, it is crucial to create an RNase-free environment. This involves thoroughly cleaning with RNA protective agents and using separate sets of equipment, as well as RNase-free consumables and reagents. Decontamination solutions such as RNaseZap, RNase AWAY, and others can be applied to laboratory equipment and work surfaces to ensure they are free of RNases. Lastly, Bahlmann recommends choosing an isolation method that is gentle on the RNA, ensures thorough lysis, and maximizes yield while efficiently removing inhibitors. This significantly reduces RNA fragmentation and decreases the probability of challenges in downstream assays.

Extraction techniques by sample types

The initial step in choosing the right extraction kit is to review the nature and characteristics of the samples. “Each input material comes with different challenges due to its unique composition and structure,” explained Bahlmann. For example, blood samples require careful handling due to their complex makeup. Researchers may need to use specific methods to remove proteins like serum albumin and anticoagulants, which help eliminate enzyme-inhibiting compounds and prevent coagulation. Additionally, cell separation is typically necessary to isolate the cells of interest, such as leukocytes.

Bahlmann also shared that efficient homogenization and lysis are often required, while mechanical disruption is commonly used for difficult-to-lyse tissues. Researchers should evaluate whether mechanical, chemical, or enzymatic lysis is most appropriate for their sample type. “On the molecular level, chemical structures can affect the success of our experiment,” Bahlmann explained. A good example is plant material, which typically requires additional steps and buffers to remove polysaccharides and phenolic compounds. Challenging sample types, such as environmental, wastewater, and formalin-fixed paraffin-embedded (FFPE) samples, are difficult to process due to their tough matrix and high levels of inhibitors, and often necessitate the use of specialized extraction kits and protocols to achieve high-quality RNA yields.

Criteria for selecting RNA extractions

After evaluating sample characteristics, there are various other factors to consider before deciding on an RNA extraction method. Despite the wide range of RNA extraction kits on the market, Bahlmann optimistically noted that choosing an RNA isolation kit is not as tricky as we have been told. To start, researchers should consider the requirements for their downstream experiments. The selected method must lead to the necessary yields and purities, while also isolating the desired RNA species (e.g., mRNA, miRNA, total RNA, etc.). Additionally, the speed of the method is important to prevent RNA degradation. Streamlined kits and those compatible with automation can simplify the workflow and minimize the risk of degradation. Bahlmann also emphasized that sustainability is a key factor, as extraction kits can vary significantly in plastic consumption and use of hazardous reagents. Finally, cost is a major consideration. "We recommend taking all costs into account, including labor, associated consumables, and waste disposal," Bahlmann advised.

Recent developments

While most available extraction methods have relied on the same chemistry since the 1980s, the good news, Bahlmann emphasized, is that there is still innovation within the field. She noted that this includes BioEcho's single-step purification technology that streamlines RNA isolation by eliminating binding, washing, and eluting steps, making workflows faster and more convenient while reducing hazardous chemicals.

Driven by scientists' ongoing efforts to refine their research, many other advancements have emerged in the field. Notably, developments in plant protocols have introduced new approaches for extracting high-quality RNA from plant tissues rich in starch, proteins, and fiber.^1,2 Significant advancements have also been made in RNA extractions from blood³, FFPE tissues⁴, and cell-free RNA⁵, among many others. Furthermore, the recent SARS-CoV-2 pandemic has prompted innovations in RNA isolation, including methods that streamline detection processes to identify RNA without the need for extractions.^6,7,8

Final advice

Along with her previous recommendations, Bahlmann offered valuable guidance for newcomers to the field. “The most important advice for a novice is to rigorously maintain an RNase-free environment and choose your isolation method wisely,” she stated. The pervasiveness of RNases and the fragile nature of RNA make it imperative to prevent contamination at all stages of the workflow. Bahlmann also reemphasized the importance of understanding the sample thoroughly, identifying the required input amount, and making necessary adaptations. Recognizing the specific characteristics of each sample type allows researchers to optimize their protocols effectively. Furthermore, Bahlmann explained how slight mistakes in technique or contamination can severely compromise RNA quality and downstream processes. Meticulous planning, careful pipetting, and strict adherence to standardized protocols are essential to avoid these pitfalls. Finally, Bahlmann advised seeking help when faced with challenges. “Before getting frustrated ask others for help. We all have some struggles from time to time.”

References

1. Vennapusa, AR, Somayanda, IM, Doherty CJ, Krishna, SV. A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber. Scientific Reports. 2020;10(1):16887. 

2. Sasi S, Krishnan S, Kodackattumannil P, et al. DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications. Plant Methods. 2023;19(1):84. 

3. Nguyen LT, Pollock CA, Saad S. Extraction of high quality and high yield RNA from frozen EDTA blood. Scientific Reports. 2024;14(1):8628. 

4. Odogwu N, Albertson S, Hagen C, Jang J, Nelson T. P852: Optimizing RNA extraction methods for high throughput transcriptome sequencing from FFPE cardiac tissue. Genetics in Medicine Open. 2024;2. 

5. Moufarrej MN, Quake SR. An inexpensive semi-automated sample processing pipeline for cell-free RNA extraction. Nature Protocols. 2023;18(9):2772-2793. 

6. Zhao Z, Cui H, Song W, Ru X, Zhou W, Yu X. A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2. bioRxiv. Published online January 1, 2020:2020.02.22.961268. 

7. Somvanshi, SB, Kharat, PB, Saraf, TS , Somwanshi, SB, Sumit, SB, Jadhav, KM. Multifunctional nano-magnetic particles assisted viral RNA-extraction protocol for potential detection of COVID-19. Materials Research Innovations. 2021;25(3):169-174. 

8. Alafeef M, Moitra P, Dighe K, Pan D. RNA-extraction-free nano-amplified colorimetric test for point-of-care clinical diagnosis of COVID-19. Nature Protocols. 2021;16(6):3141-3162.