Exploring the impact of primer length on efficient gene detection via high-throughput sequencing.

Reverse 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.