Researchers are continuously seeking ways to improve efficiency while ensuring that their methods are reliable and their research is reproducible. Replicating experimental results has proven to be difficult,1,2 and research tools play a key role.3 While emerging technologies, such as next-generation sequencing, have increased throughput, they require sample-processing tools that are able to keep pace. However, basic sample-processing tools and techniques critical to obtaining reliable, accurate results are often overlooked.
A routine procedure that can be optimized is repetitive pipetting—the sequential dispensing of equal-volume aliquots. Switching from mechanical to electronic pipettes—which allow for semiautomated dispensing of liquids—can increase throughput and provide greater precision, even among different users. A motor reduces the force required to operate the pipette, reducing the repetitive strain injuries and mental fatigue that can be caused by lengthy pipetting sessions.
Electronic pipetting
Electronic pipettes do not always consistently produce accurate aliquot volumes. Markedly different results were obtained when the same operator performed repetitive pipetting of ten 10-μL aliquots using electronic pipettes from different manufacturers (see Figure 1).
Figure 1 – Aliquot volume variation using electronic pipettes from different manufacturers. Manufacturer 1: 300-μL single-channel pipette tested with manufacturer tips (red). Manufacturer 2: PIPETMAN M P300M: 300-μL single-channel pipette tested with manufacturer tips (teal). The permissible errors noted on the graph represent volumetric guidelines set forth by ISO for standard pipetting mode. Measurements follow ISO Standard 8655-6, “Gravimetric method for the determination of measurement error.”Keys to accurate repetitive pipetting
To maximize reproducibility of results derived from repetitive pipetting, it is most important to select a high-quality air-displacement electronic pipette. Ideally, the pipette should be paired with tips from the same manufacturer.
Understanding the mechanics of and best practices for repetitive pipetting is crucial when selecting the ideal electronic pipette and tip pair.
In standard pipetting, the intended volume is aspirated and fully dispensed once per sample. During repetitive pipetting, the intended volume is dispensed multiple times (Figure 2) by aspirating a large volume that is divided into many small aliquots. This reduces time and effort. The pipette must be able to deliver an accurate volume for each aliquot, which can be challenging using a nonadapted pipetting technique.
Figure 2 – Schematic of steps for repetitive pipetting mode.Although the various manufacturers of electronic pipettes employ different mechanisms, several factors are universally important to ensure accuracy and precision. These include adjustment of the motor between aliquots, calculation of the appropriate amount of aspirated volume, and removal of necessary extra volume during preparation for aliquoting. Selecting a pipette that has had each parameter carefully set and optimized is critical for achieving consistency and the best results.
Electronic pipette specifications
Permissible levels of systematic and random error for standard pipetting with air displacement are set by the International Organization for Standardization (ISO) 8655-2. Pipette precision is represented by random error and refers to the deviation around the intended volume. Systematic error, on the other hand, is the difference between the measured and actual dispensed volume, and represents the pipette’s accuracy.
Although electronic pipettes are widely used for repetitive pipetting in biological assays, there is no ISO standard for repetitive pipetting using air-displacement pipettes, and manufacturers typically do not provide information on the volumetric specifications for the repetitive mode of these devices. PIPETMAN M electronic pipettes from Gilson (Middleton, Wis., U.S.A./Villiers-le-bel, France) use ISO specifications for standard pipetting as a reference for ALestablishing volumetric specification for their repetitive pipetting mode.
Proper technique ensures pipetting accuracy
Electronic pipetting has the potential to provide greatly increased user-to-user and aliquot-to-aliquot consistency, and optimal results require proper pipetting technique. To start, the electronic pipette should be held nearly vertical while the solution is aspirated. Excess volume should be discarded into the source vessel in preparation for dispensing the first aliquot. To dispense, the electronic pipette is held with the tip against the wall at less than a 45-degree angle. Wiping the tip against the wall of the vessel is recommended to ensure that any liquid drop remaining at the end of the tip is dispensed.
Proper pipetting technique has a clear impact on the consistency of aliquot dispensing. In one experiment, a technician pipetted ten 20-μL aliquots before and after training (Figure 3). Prior to training, the technician dispensed a number of aliquots outside of the range of permissible error, as well as a more general deviation around the intended volume (even for those in the permissible range). Proper technique following training resulted in aliquots with greater accuracy and less variation in volume. Given the importance of proper pipetting technique, researchers should regularly review and refresh their knowledge for optimal results, and should consider taking advantage of training sessions offered by pipette manufacturers.
Figure 3 – The effects of proper pipetting technique on 10 dispensed aliquots using a PIPETMAN M P200M pipette and PIPETMAN DIAMOND tips. Results from before (orange curve) and after (green curve) training are shown. ISO guidelines for standard pipetting mode were used to determine permissible errors shown on the graph. Measurements follow ISO Standard 8655-6, “Gravimetric method for the determination of measurement error.”Conclusion
While the principles of repetitive pipetting are standard, execution is not. Selection of the correct pipette can significantly impact performance. Proper pipetting technique is also critical to ensure reliable results—poor technique is detrimental to accuracy and precision and can result in experimental errors. Electronic pipettes with repetitive pipetting capabilities enable reliable sample preparation.
References
- Baker, M. Biotech giant publishes failures to confirm high-profile science. Nature 2016, 530(7589), 141; doi:10.1038/nature.2016.19269.
- Mullard, A. Reliability of ‘new drug target’ claims called into question. Nat. Rev. Drug Discov. 2011, 10(9), 643–4; doi:10.1038/nrd3545.
- Freedman, L.P., Cockburn, I.M. et al. The economics of reproducibility in preclinical research. PLOS Biol. 2015 13(6); doi:10.1371/journal.pbio.1002165.
Nora Meneceur is product manager, and Beatrice Guieu-Presle is test laboratory manager, Gilson, 19 avenue des entrepreneurs, 95400 Villiers-le-bel, France; tel.: + 00 33 1 34 29 50 00; e-mail: [email protected]; www.gilson.com