Experimental Reproducibility: How to Get the Most “Bang” for Your Buck

As scientists, we are trained to design an experiment with the bigger picture in mind; the ultimate goals being to publish quality data and demonstrate scientific rigor. However, sometimes you need to focus on the little things, such as perfecting control and experimental samples, incubation times, and ordering reagents to truly ensure that you obtain reproducible results. You also must account for those not-so-obvious variables, like consistency of reagent lots, environmental conditions and reliable consumables. If you take the time to verify experimental controls, reagents, equipment, and environmental parameters you will strengthen the repeatability and reproducibility of your data. Here are some suggestions to ensure you are doing everything in your power to obtain reproducible and reliable data.

Experimental Set-Up

1. Pay Attention to Controls

As cumbersome as it may be, take the time to plan the details of your experiment. In particular, you should pay attention to the development or plan of appropriate experimental controls. Without the complete set of controls in place, your data will be uninterpretable, and you will need to run your experiment again.

2. Simplify When at All Possible

Large experiments promise to deliver a lot of data, which is exciting. They can also induce stress, consume all your time, and lead to unfortunate mistakes because of their intricate construction. To avoid these possible issues, refine your experiment so that you can handle it comfortably—without stress. If possible, design it to yield data in an easily interpretable format.

3. Check Your Equipment

Ensure that the laboratory equipment is in working order. This is especially true for a shared piece of equipment. Ideally, you would check and validate the instrument during the experimental design phase to ensure reliability. Following maintenance and calibration, run a pilot experiment with control samples to verify that the machine works properly under experimental conditions.

Maintaining Consistency

1. Detailed Protocols

Having a detailed written protocol will help you to maintain consistency and replicate your own results, but it also allows others in your lab to use the same experimental setup with confidence. To ensure that your written instructions are detailed enough, check its inter-observer reliability. You can do this by asking a labmate to perform the protocol and note down anything that they feel is unclear.

2. Reagents

Despite tightly regulated quality control measures during the production of reagents and consumables, slight variations occur from time to time that can have profound effects on downstream results. Therefore, you must be consistent with your chosen reagents. That is, you should order the same reagents from the same suppliers every time.

This will require you to monitor the amount of reagent remaining so that you can order more before it runs out. Keep a close eye on lot numbers when ordering and document changes in lot numbers as they occur. While documentation of lot numbers may not be necessary for common items, such as PBS or distilled water, including this information in your records can be useful for developing good habits and on the off chance that your experiment is the basis of a clinical trial.

Changing reagent lots in the middle of a long-term experiment is sometimes unavoidable. When this happens, you should test the new reagent (from the new lot) for efficacy. This step may cost you time and may seem excessive, but accounting for possible differences between lots is essential to avoid or at least minimize future troubleshooting.

3. Consumables

Items such as flasks, serological pipets, and pipette tips may seem innocuous, but variability between lots can lead to suboptimal culture environments or incorrect pipetting volumes. For example, with pipette tips, using low retention tips is advisable. These tips dispense the entire sample easily, without collecting inside the tip. Moreover, tips that improve sample uniformity and CV (coefficient of variation) values, such as those available at Biotix, diminish inconsistencies with volume. Therefore, low retention tips increase the precision and robustness of your data.

4. Environmental Parameters

Environmental parameters to consider include the pH of buffers and reagents and the incubation temperature(s) required during experimental steps. Assuming the pH of a reagent based on the label can be tempting (especially if you have purchased it directly from a company), but it is ultimately your responsibility to verify this before use. Similarly, if a step in an experiment calls for incubation at room temperature, ensure that your lab is not too hot or too cold because of seasonal adjustments in your building’s thermostat. To be on the safe side, carry out room temperature incubations in an incubator set to 25 °C.

More Reproducibility, Less Troubleshooting!

While the above tips and suggestions should help you to achieve reproducibility and avoid troubleshooting, you will probably find yourself with a set of completely unreliable data at some point or another. This is frustrating, but it may help to bear in mind that reproducibility can be influenced by factors completely beyond your control e.g., unforeseen changes in building temperature, or factors that may seem insignificant e.g., the choice of pipette tips. If you find yourself in this situation, think outside the box, because paying attention to the least obvious sources of error, such as pipette tips, might help. Importantly, by following our tips, the list of what could have gone wrong will be significantly reduced, saving you time and money in the long run.