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Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli
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Cost-Effective Method for Using Cross-Species Spike-In RNA for Normalization and Quantification in Polysome Profiling

Krishna Bhattarai1, Angelo Slade1, Martin Holcik1

  • 1Department of Health Science, Carleton University, Ottawa, ON K1S 5B6, Canada.

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|November 27, 2025
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Summary

Cross-species total RNA, like yeast RNA, serves as a cost-effective spike-in control for RNA quantification in experiments such as RT-qPCR. This method ensures reliable results without interfering with endogenous RNA measurements.

Keywords:
Bcl-xLhypertonic stresspolysome profilingspike-in RNA control

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • Accurate RNA quantification is essential for molecular biology techniques like RT-qPCR and polysome profiling.
  • Conventional spike-in controls (in vitro mRNA, ERCC mixes) are costly and time-consuming, limiting their accessibility.
  • There is a need for economical and reliable RNA controls in experimental settings.

Purpose of the Study:

  • To evaluate the efficacy of cross-species total RNA as a cost-effective spike-in control.
  • To assess the impact of yeast RNA as a spike-in on RNA quantification accuracy and reproducibility in human cell experiments.
  • To validate the method in a specific application, such as assessing mRNA translation efficiency under stress.

Main Methods:

  • Developed a method utilizing yeast total RNA as a spike-in control for human cell-based RNA assays.
  • Tested the yeast RNA spike-in across various RNA-based experiments, including RT-qPCR and polysome profiling.
  • Applied the method to investigate Bcl-xL mRNA translation efficiency during hypertonic stress.

Main Results:

  • Cross-species spike-in RNA showed minimal interference with experimental results and provided consistent normalization.
  • Yeast RNA spike-in enabled accurate fold-change calculations and better detection of experimental variability.
  • The spike-in control facilitated reliable assessment of Bcl-xL mRNA translation efficiency under stress conditions.

Conclusions:

  • Total RNA from a non-related species presents a practical and economical alternative to traditional spike-in controls.
  • This approach improves RNA quantification reliability without compromising experimental integrity.
  • The method is particularly beneficial for resource-limited laboratories and essential for polysome and RT-qPCR workflows.