LABTips: Life Science Sample Prep

Sample preparation can be a bottleneck in the life sciences lab, and errors during this crucial task can be detrimental to your downstream analyses. For this reason, finding ways to increase the efficiency and accuracy of processes like liquid dispensing and nucleic acid extraction is the first step toward increasing your throughput and the repeatability of your results. Consider the following tips for both optimizing your current methods and exploring new methods for streamlined and consistent sample preparation: 

1. The Smaller the Better

Miniaturizing assays has many benefits, perhaps the most obvious being the ability to save costs and reduce waste by using less reagent per analysis. Miniaturization also uses less sample, and provides the opportunity to obtain more data from a single sample through multiple tests. Scaling down your reaction volume can also scale up your throughput by allowing you to run more tests simultaneously — up to a four-fold increase when switching from a 96-well plate to a 384-well plate, for example.¹ 

Assay miniaturization has been applied to COVID-19 research in areas of both drug discovery and diagnostics. Miniaturizing assays is one way to conserve reagent amid supply chain disruptions and shortages.² High-throughput screening of existing drug libraries using high-density microplates and just a few microliters of reagent per well has also allowed researchers to mine through thousands of candidates in much fewer runs than a 96-well format would allow.³ Additionally, miniaturization has benefits for RT-PCR testing, having the ability to both increase testing capacity and reduce costs. 

In order to fully reap the benefits of miniaturization, be aware of some of the challenges associated with denser microplates and smaller reaction volumes. Plates with a larger amount of smaller wells will be more sensitive to interference from dust and air bubbles.⁴ Plate centrifugation and sonic disruption are two methods that can help eliminate bubbles, and dust and other sources of contamination can be avoided by maintaining a clean work environment, as well as inspecting your plate for foreign materials prior to use. Adjusting your workflow to get ahead of these challenges will ensure your high-throughput processes run smoothly. 

2. Control Evaporation

Reagent evaporation can impact results and reduce the repeatability of your experiments. Like dust and air bubbles, evaporation becomes an even greater concern when working with much smaller reaction volumes on miniaturized microplates (eg. 384-well, 1538-well or higher).⁴ One of the simpler, but less precise, ways to mitigate evaporation is by filling the perimeter wells of a microplate with water or another medium to reduce the “edge effect,” and some microplates come with extra “dummy wells” specifically for this purpose so you don’t lose access to any of your assay wells.

Another simple method for evaporation control is covering your plate with adhesive microplate-sealing tapes or films. However, you’ll want to be sure the seal you use is compatible with your application; for example, make sure the seal can withstand autoclaving or extremely low temperatures if necessary. You’ll also want to take care to avoid cross-contamination when removing or piercing the seal. 

Because environmental factors like relative humidity and temperature can greatly affect evaporation rate, monitoring changes in environmental conditions will be important for understanding how evaporation may affect your experiment. Conducting your experiments in humidity-controlled chambers or rooms can significantly reduce these environmental effects. Automated systems can also help monitor and control these factors, and can also handle tasks like seal placement and removal for evaporation control in high-throughput operations. 

3. Be Ready for Next-generation Sequencing

Next-generation sequencing, or NGS, technologies have greatly accelerated the study of genomics, yet life science labs can still hit bottlenecks due to laborious manual sample preparation. Thus, streamlining sample prep is a vital step in unleashing the full potential of this revolutionary technology.⁵ Automation is the most straightforward solution to increasing throughput and reducing human error that might result in reruns, but this does not mean you must immediately go out and buy an expensive, sophisticated all-in-one robotic NGS workstation. 

Semi-automation through single-tasking instruments, like reagent dispensers, microplate sealers and labeling systems, can provide an efficiency boost by freeing up hands that would otherwise be tied up performing tedious, repetitive tasks. Automating workflow steps for which human error is most likely to impact consistency and accuracy — through imprecise pipetting, for example — is one smart way to avoid reruns and improve repeatability.⁶ 

Another step that can consume a lot of time and become a source for errors is DNA extraction, especially when working with high molecular weight (HMW) DNA for long-read sequencing. Phenol-chloroform based extraction methods, while reliable and relatively inexpensive, are incredibly time-consuming and involve hazardous chemicals. If you’re looking to streamline your DNA extraction, you may want to explore different techniques and protocols, which may include different reagents or different formats such as magnetic bead extraction or column-based extraction.⁷ 

For HMW DNA extraction, preventing shearing in every step from lysis to storage is key to ensuring nice long reads for downstream sequencing. Minimize harsh physical and chemical processes that could damage HMW DNA wherever possible, and take proper precautions to prevent degradation from DNase or improper temperature conditions.⁸

Tip 4: Take Less Common Techniques into Account

Liquid-liquid extraction and solid phase extraction are still the most popular sample preparation techniques. However, use of other methods, including supported liquid extraction (SLE), are on the rise.

SLE allows unwanted materials in a biological sample to be retained on a solid surface comprising natural materials, such as silica. There are typically three steps involved in the SLE process: (1) loading the column, (2) waiting while the matrix is absorbed into the column, and (3) eluting the analytes of interest. Increased reproducibility, analyte recovery and reduction in ion suppression have all been demonstrated with the use of SLE. SLE methods give cleaner extracts compared with liquid-liquid techniques due to phospholipids and other matrix interferences binding to the column and not co-eluting.⁹

If you work in a forensic laboratory processing toxicological samples—or a biological lab that tests for drugs of abuse—you may find SLE methods to be especially useful. Previous studies have shown SLE is efficient and effective at extracting tetrahydrocannabinol (THC) and metabolites from whole blood and oral fluid for LC-MS/MS analysis. The technique also easily extracts opiates and benzodiazepines from whole blood, and can extract a typical drugs-of-abuse panel from oral fluid.

5. Consider Non-contact Dispensing

While pipettes are ubiquitous in the life science lab, both in manual and automated formats, pipetting is not the only option for liquid handling and dispensing. Automated non-contact, non-tip-based dispensing can help avoid contamination and carryover without burning through pipette tips, thus reducing both consumable costs and plastic waste.¹⁰ These alternative dispensing systems can also help preserve reagent due to much lower dead volume than traditional contact methods.¹¹ 

As digital and automated systems, non-contact dispensers avoid the disadvantages of manual pipetting and can be programmed to dispense precise droplets in microliter, nanoliter or even sub-nanoliter volumes. This is useful for miniaturized assays, and in addition, contactless systems can eliminate the step of manual serial dilution by automatically dosing individual wells with different volumes. 

A few things to keep in mind when considering a switch to a non-contact system are the need for separate mixing/shaking to replace pipette mixing, the system’s workflow for handling multiple reagents and which method of non-contact dispensing is right for your application (e.g. acoustic vs. inkjet formats). 

While no method is one-size-fits-all, knowing the different options and their potential to optimize workflow steps for your samples and application will provide the opportunity to reach for better and faster sample preparation.

References

1. “The Why and How of Miniaturizing Genomics Applications,” SPT Labtech. https://discovery.sptlabtech.com/miniaturization-genomics-ngs-pcr-complete-guide
2. “Automation and Miniaturization Solution to Testing and Research Bottlenecks,” SPT Labtech. https://www.sptlabtech.com/whats-new/news/solution-to-testing-and-research-bottlenecks/
3. Smith E, Davis-Gardner ME, Garcia-Ordonez RD, et al. High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2. SLAS DISCOVERY: Advancing the Science of Drug Discovery. 2020;25(10):1152-1161. doi:10.1177/2472555220963667
4. Kong F, Yuan L, Zheng YF, Chen W. Automatic Liquid Handling for Life Science: A Critical Review of the Current State of the Art. Journal of Laboratory Automation. 2012;17(3):169-185. doi:10.1177/2211068211435302
5. Tegally, H., San, J.E., Giandhari, J. et al. Unlocking the efficiency of genomics laboratories with robotic liquid-handling. BMC Genomics 21, 729 (2020). https://doi.org/10.1186/s12864-020-07137-1
6. “Expert Tips to Improve NGS Sample Prep,” Biocompare. https://www.biocompare.com/Editorial-Articles/574058-Expert-Tips-to-Improve-NGS-Sample-Prep/ 
7. Psifidi A, Dovas CI, Bramis G, Lazou T, Russel CL, Arsenos G, et al. (2015) Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples. PLoS ONE 10(1): e0115960. https://doi.org/10.1371/journal.pone.0115960
8. “Sequencing 101: DNA Extraction – Tips, Kits, & Protocols,” PacBio. https://www.pacb.com/blog/sequencing-101-dna-extraction/  
9. Jones, S., McGowan, C., Boyle, S., Ke, Y., Chan, C. H. M., & Hwang, H. (2021). Anoverview of sample preparation in forensic toxicology. Wiley Interdisciplinary Reviews: Forensic Science, e1436. https://doi.org/10.1002/wfs2.1436
10. “Thermo Scientific Multidrop Pico 1 and 8 Digital Dispenser,” Thermo Fisher Scientific. https://assets.thermofisher.com/TFS-Assets/LPD/Product-Information/LPD-LPE-Multidrop-Pico1and8-Brochure.pdf 
11. “Multidrop Combi Reagent Dispenser Workflow Solutions,” Thermo Fisher Scientific. https://assets.thermofisher.com/TFS-Assets/LED/Technical-Notes/Multidrop_Combi_DispenserWorkflowSolutions.pdf