Sample Processing for Nucleic Acid Analyses

Nucleic acids (NAs) contain genetic information that comprises an organism’s phenotype. Its foundational role in driving the functions of living cells has made DNA and RNA profiling a staple in biomedical research. However, profiling these NAs requires a robust sample processing protocol centered around their extraction from biological material. “NA extraction is an essential yet routine procedure for many downstream applications in molecular biology, from gene editing to drug discovery,” explains Anna C. Lai, Global Product Manager of PCR Reagents at Bio-Rad. Therefore, scientists must integrate a robust sample processing protocol into their workflows to advance their biomedical research.

What is sample processing?

Sample processing encompasses a series of steps that isolate NAs from diverse sample matrices. These workflows encompass protocols that enrich specific cell types, remove contaminants, and extract NAs from the samples. Obtaining pure NAs for downstream applications represents the primary goal of any sample processing protocol.

Factors affecting sample processing efficiency

Sample processing pipelines take many forms. “The best sample processing protocol depends on multiple variables,” Lai adds. “The type and quality of the starting sample, the target NA species, and the downstream application may all affect which protocol will work best.”

Constantine Garagounis, Product Development & Marketing Specialist at PCR Biosystems, expounds on the nature of the starting sample as a variable further. “The presence of certain contaminants in the NA can produce unreliable results in downstream applications. These include compounds that inhibit reactions, NA crosslinking agents that reduce PCR amplification, and residual chemicals from extraction reagents. Minimizing sample contaminants like these will maximize the chances of success in a downstream reaction.”

Different NA species will also require distinct extraction protocols. “Plasmid DNA extractions require that you separate it from contaminating genomic DNA,” Lai notes. “Gentler lysis methods also ensure that the plasmid DNA remains intact without signs of shearing. For RNA extraction, RNAses—enzymes that degrade RNA—are present everywhere and can compromise your sample. Researchers must inactivate endogenous RNAses in all laboratory equipment, work surfaces, and consumables. On top of that, selecting the right RNA isolation method ensures that you obtain intact RNA for your downstream experiments.” These considerations have led scientists to create a metric for measuring RNA integrity of isolated RNA, which is called the RNA integrity number (RIN).¹

Scientists must carefully consider multiple variables to optimize their sample processing protocols. Kits that account for these variables will help streamline the sample processing protocol and produce robust NA extracts. For example, Lai says of  Bio-Rad’s line of kits, “First, the Aurum Plasmid Mini Kit can deliver high-purity plasmid DNA in 15 minutes. It is designed to be used in both spin and vacuum formats (compatible with Aurum vacuum manifold). Our silica membrane technology has a strong binding affinity to plasmid DNA, allowing easy DNA isolation. We also sell an array of RNA isolation reagents ranging from Chelex 100 and PureZOL for addressing all your RNA extraction needs.” 

Applications in sample processing protocols

Sample processing protocols occupy the first step in any successful NA profiling pipeline. For one, generating robust sequencing data requires an extraction protocol that keeps NAs intact. This ensures that NAs can be fragmented into the correct sizes when preparing sequencing libraries.² Obtaining pure NAs also improves the chances of a successful PCR experiment.

Aware of this need, Garagounis recommends PCR Biosystems' Rapid Extract PCR Kit for three specific reasons. “First, the kit is easy to use. It’s a simple protocol that on average only requires 15-20 minutes to complete and only has a few steps. The kit is also suitable for many kinds of soft tissues, from blood to hair follicles, and even Formalin-Fixed Paraffin-Embedded (FFPE) samples. Finally, we don’t use any hazardous phenols or strong alkaline solutions during our preparations, nor do we have a washing step. Altogether, our kit provides substantial amounts of high-quality NAs in less than an hour. This allows our users to increase workflow efficiency and experimental reproducibility.”

Automation in sample processing protocols

Technical advances in molecular biology, such as high-throughput sequencing, have raised the need for processing many samples quickly. This effort also requires researchers to ensure reproducibility by maintaining precision and accuracy. As Elvy Fan, Senior Product Specialist of Chroma ATE, points out, “robust molecular biology research requires that we rely on rigorous technical precision and accuracy. This helps us ensure that no matter how many times we repeat the experiment under the same conditions, that we can obtain the same results.”

This need has pushed forward efforts to develop automated pipetting systems. Arise Biotech has helped meet this need with their EzMate Automated Pipetting System. Jeffrey Lai, Product Manager at Arise Biotech, explains that “the pipetting system can be equipped with a multi-channel 50 µL or 200 µL automated pipetting module. When mounted on a robotic arm, the system allows for precise pipetting of microliter volumes to facilitate your sample processing needs. The system is also housed within a cabinet, equipped with a HEPA intake air filtration system, and installed with a UV lamp to keep the inner environment clean and sterile.”

Chroma ATE has also developed their own automated system for processing nucleic acids. The MagXtract 3200 “comprises a number of features that make processing many samples for genetics-based applications easy,” says Fan. She continues, stating that the MagXtract 3200 “is designed with the patented whir-and-stir approach to maximize sample mixing efficiency. The machine is also equipped with a high-quality air displacement pipettor to achieve precise and accurate liquid handling. Finally, the MagXtract 3200 hosts UV-C light and HEPA filters to decontaminate the machine before and after experiments.”

With companies like Arise Biotech and Chroma ATE developing automated liquid-handling systems, sample processing pipelines will become that much more reproducible, reliable, and robust.

Conclusion

Every successful NA study requires a way to extract pure NAs. Many factors impact the quantity and quality of the NA extracts obtained from sample processing. From storage conditions to the reagents employed, optimizing each step of a sample processing protocol will help produce robust data for downstream applications. A wide array of extraction kits and reagents is available to make this process easier. Moreover, advances in automation will streamline these protocols by automating repetitive volume transfer tasks. With many kits and automated systems available, sample processing pipelines have a bright future, providing a platform to take NA research to the next level.

References

1. Schroeder A, Mueller O, Stocker S, et al. The RIN: An RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. Published online 2006. doi:10.1186/1471-2199-7-3

2. Hess JF, Kohl TA, Kotrová M, et al. Library preparation for next-generation sequencing: A review of automation strategies. Biotechnology Advances. 2020;41:107537. doi:10.1016/j.biotechadv.2020.107537