Abstract
AbstractReverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.
Publisher
Springer Science and Business Media LLC
Reference46 articles.
1. Bustin, S. et al. Variability of the reverse transcription step: practical implications. Clin. Chem. 61, 202–212 (2015).
2. Schwaber, J., Andersen, S. & Nielsen, L. Shedding light: The importance of reverse transcription efficiency standards in data interpretation. Biomol. Detect. Quantif. 17, 100077 (2019).
3. Bustin, S. A. & Nolan, T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. JBT 15, 155–166 (2004).
4. Lindén, J., Ranta, J. & Pohjanvirta, R. Bayesian modeling of reproducibility and robustness of RNA reverse transcription and quantitative real-time polymerase chain reaction. Anal. Biochem. 428, 81–91 (2012).
5. Ståhlberg, A., Håkansson, J., Xian, X., Semb, H. & Kubista, M. Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50, 509–515 (2004).