Cost-Effective Method for Using Cross-Species Spike-In RNA for Normalization and Quantification in Polysome Profiling Experiments

Background/Objective: Accurate quantification of RNA is critical for RNA-based experiments such as polysome profiling and RT-qPCR. These techniques often rely on control RNA to ensure consistency and reliability across experiments. Commonly used spike-in controls, including in vitro-synthesized mRNA or ERCC mixes, are expensive and time-consuming, limiting accessibility for many laboratories. This study aims to evaluate the use of cross-species total RNA as a cost-effective and reliable spike-in control. Methods: We developed a method using total RNA from a non-homologous species—specifically, yeast RNA—as a spike-in control for experiments involving human cells. The approach was tested across multiple RNA-based assays to assess its impact on quantification accuracy, reproducibility, and interference with endogenous RNA measurements. Additionally, we applied this method to evaluate the translation efficiency of Bcl-xL mRNA in mammalian cells under hypertonic stress. Results: Cross-species spike-in RNA demonstrated minimal interference with experimental outcomes and provided consistent normalization across replicates. The use of yeast RNA enabled accurate fold-change calculations and improved detection of experimental variability. In the case study involving Bcl-xL mRNA, the spike-in control facilitated reliable assessment of translation efficiency under stress conditions. Conclusions: Using total RNA from a non-related species as a spike-in control offers a practical, economical alternative to conventional methods. This approach enhances the reliability of RNA quantification without compromising experimental integrity, making it especially valuable for resource-limited settings, particularly in polysome and RT-qPCR workflows.