Optimizing Small RNA Sequencing for Salivary Biomarker Identification: A Comparative Study of Library Preparation...

Area of Science:

• Molecular Biology
• Genomics
• Biomarker Discovery

Background:

• MicroRNAs (miRNAs) are key gene regulators with diagnostic potential.
• Saliva offers a non-invasive source for miRNA biomarker analysis.
• Next-generation sequencing (NGS) miRNA profiling is sensitive to library preparation methods.

Purpose of the Study:

• To compare four commercial small RNA library preparation kits for miRNA profiling.
• To evaluate kit performance using cell-free saliva, plasma, and extracellular vesicles (EVs).
• To assess efficiency, bias, and miRNA detection across different sample types and kits.

Main Methods:

• Comparative analysis of four small RNA library preparation kits (QIASeq, RealSeq, Lexogen, NEBNext).
• Profiling of miRNAs from cell-free saliva, plasma, and their EVs.
• Evaluation using synthetic and biological samples, focusing on low RNA input, bias, and detection breadth.

Main Results:

• QIASeq exhibited the highest miRNA mapping rates and broadest miRNA detection, especially in saliva.
• QIASeq demonstrated minimal adapter dimer formation compared to other kits.
• Significant overlap in detected miRNAs between saliva and plasma supports saliva's utility.

Conclusions:

• Library preparation protocol significantly impacts miRNA sequencing results.
• The QIASeq kit is recommended for robust miRNA profiling from non-invasive samples like saliva.
• This study provides guidance for selecting optimal protocols for miRNA biomarker discovery.