Understanding ERCC Controls in RNA-seq Experiments
In RNA-seq experiments, External RNA Controls Consortium (ERCC) spike-in controls play a crucial role in assessing assay performance, monitoring technical variability, and quantifying gene expression. Among these controls, two commonly discussed types are ERCC ExFold and ERCC RNA Spike-In Mix. Let’s delve into their differences, advantages, and considerations for usage.
ERCC ExFold (with Mix1 and Mix2)
Purpose
ERCC ExFold comprises a set of spike-ins tailored for fold-change experiments. This mix consists of 92 transcripts, each at a known concentration, spanning a broad dynamic range.
Pros
- Ideal for assessing fold-change accuracy.
- Offers known concentration levels to evaluate assay precision across a wide dynamic range.
- Valuable for benchmarking and quality control in differential gene expression studies.
Cons
- May not suit experiments primarily focused on absolute quantification, as it emphasizes relative fold changes.
ERCC RNA Spike-In Mix (with only Mix1)
Purpose
The ERCC RNA Spike-In Mix includes 92 synthetic RNA molecules, each at a known concentration, primarily for absolute quantification experiments.
Pros
- Essential for accurately quantifying absolute gene expression levels.
- Useful for estimating RNA molecule abundance, aiding biomarker discovery.
Cons
- Less informative for fold-change experiments, prioritizing absolute quantification.
In summary, the choice between ERCC ExFold and ERCC RNA Spike-In Mix hinges on the experiment’s objectives. For assessing fold-change accuracy, ERCC ExFold is preferable, while ERCC RNA Spike-In Mix suits studies focused on absolute quantification.
It’s noteworthy that both ERCC control types can coexist in an experiment to evaluate both fold-change accuracy and absolute quantification. However, careful planning is essential to avoid complexity and increased costs.
Conclusion
- No ERCC: Often unnecessary if detecting differentially expressed genes (DEGs) without requiring absolute measurements.
- With ERCC RNA Spike-In Mix: Beneficial when expecting genome-wide overexpression or knockdown effects.
- With ERCC ExFold: Ideal for measuring fold changes in low-expressed genes, enhancing confidence without calibration.
- When using only Mix1, ERCC controls serve as negative controls for absolute measurements. Incorporating both Mix1 and Mix2 provides a positive control, bolstering confidence in fold-change accuracy rather than calibrating measurements.
By understanding the nuances of ERCC controls, researchers can make informed decisions to optimize RNA-seq experiments for their specific objectives.