spikein

Spike-in refers to defined quantities of exogenous molecules added to a biological sample to serve as internal standards or calibration references in analytical assays. The added material is designed to be distinguishable from endogenous molecules so it can be measured separately without altering the biological properties of the sample.

Spike-in controls are widely used to assess technical performance, enable normalization, and calibrate quantitative measurements in molecular biology workflows such as RNA sequencing, quantitative PCR, proteomics, and metabolomics.

In RNA sequencing, synthetic RNA transcripts or other oligonucleotides are added in known amounts before library preparation. These external controls provide a reference for the dynamic range, detection limits, and cross-sample normalization of read counts, helping to distinguish technical variability from biological differences.

A well-known example is the External RNA Controls Consortium (ERCC) spike-in set, a collection of transcripts with predefined concentrations that are used to assess RNA-seq performance across different experiments.

In proteomics and metabolomics, spike-ins often consist of stable isotope-labeled proteins or peptides added prior to sample processing. They enable absolute quantification and help monitor instrument performance and sample handling.

Considerations include the need for the spike-ins to mimic the behavior of targets to some extent, potential differences in chemistry or amplification, and careful selection of concentrations to cover the experiment's dynamic range. Spike-in-based normalization complements, rather than replaces, biological normalization strategies.