Include Both Biological and Technical Replicates in Your Experiments

Types of Replicates: Technical vs. Biological


biological replicates,technical replicates,quantification,reproducibility
“Authors must state the number of independent samples (biological replicates) and the number of replicate samples (technical replicates) and report how many times each experiment was replicated.”[1] 

When it comes to quantification, biological and technical replicates are key to generating accurate, reliable results. While they both offer researchers valuable data, each answers distinct questions about data reproducibility. Let's take a moment to explore the differences between technical and biological replicates.

What Are Technical Replicates?

Technical replicates are repeated measurements of the same sample that demonstrate the variability of the protocol. Technical replicates are important because they address the reproducibility of the assay or technique; however, they do not address the reproducibility of the effect or event that you are studying. Rather, they indicate whether your measurements are scientifically robust or noisy and how large the measured effect must be in order to stand out above the background noise.[2] Examples may include loading multiple lanes with each sample on the same blot, running multiple blots in parallel, or repeating the blot with the same samples on different days.

Figure 1. Technical replicates help identify variation in technique. For example, lysate derived from a mouse that is loaded three times (A1, A2, A3) on each membrane then run and measured independently will help identify variation in technique.

When technical replicates are highly variable, it is more difficult to separate the observed effect from the assay variation. You may need to identify and reduce sources of error in your protocol to increase the precision of your assay. Technical replicates do not address the biological relevance of the results.

What Are Biological Replicates?

Biological replicates are parallel measurements of biologically distinct samples that capture random biological variation, which can be a subject of study or a source of noise itself.[3] Biological replicates are important because they address how widely your experimental results can be generalized. They indicate if an experimental effect is sustainable under a different set of biological variables.

For example, common biological replicates include repeating a particular assay with independently generated samples or samples derived from various cell types, tissue types, or organisms to see if similar results can be observed. Examples include analysis of samples from multiple mice rather than a single mouse, or from multiple batches of independently cultured and treated cells.

Figure 2. Biological replicates derived from independent samples help capture random biological variation. For example, three biological replicates (A, B, and C) are collected from three independent mice. Each of these biological replicates was run in three technical replicates (A1, A2, A3; B1, B2, B3; C1, C2, C3) in one Western blot assay.

To demonstrate the same effect in a different experimental context, the experiment might be repeated in multiple cell lines, in related cell types or tissues, or with other biological systems. An appropriate replication strategy should be developed for each experimental context. Several recent papers discuss considerations for choosing technical and biological replicates.[1,2,3]

For a helpful guide in choosing and incorporating the right technical and biological replicates for your experiment, check out the Quantitative Western Blot Analysis with Replicate Samples protocol.

Biological vs. Technical Replicates

Proper Western blot quantification requires the keen understanding and application of both biological and technical replicate best practices. Here are some additional resources that you can use as you plan your quantitative Western blot strategy.

If you ever have questions or concerns about biological vs. technical replicates, then we hope you'll reach out for more information. Our goal is to be your constant in Western blot quantification, and we're always eager to support your lab's research however we can.

References:

  1. [1] Instructions for Authors. The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 31 July 2017.
  2. [2] Naegle K, Gough NR, Yaffe MB. Criteria for biological reproducibility: what does “n” mean? Sci Signal. 8 (371): fs7 (2015).
  3. [3] Blainey P, Krzywinski M, and Altman N. (2014) Points of Significance: Replication. Nature Methods 11(9): 879-880. doi:10.1038/nmeth.30
  4. [4] Collecting and Presenting Data. The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 9 May 2018.

Powered by Froala Editor