miRNA Library Preparation Optimisation for Low-Concentration and Low-Volume Paediatric Plasma Samples

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Analyzing circulating microRNAs (miRNAs) in pediatric plasma is difficult due to limited sample volumes.
• The QIAseq miRNA UDI Library Kit was chosen for its suitability with plasma and serum samples.

Purpose of the Study:

• To optimize the QIAseq miRNA UDI Library Kit protocol for low-volume pediatric plasma samples.
• To achieve acceptable miRNA library yields from challenging pediatric plasma samples.

Main Methods:

• Compared miRNeasy Serum/Plasma and MagMAX miRVana kits using 100 µL and 200 µL pediatric plasma.
• Prepared libraries with the QIAseq miRNA UDI Library Kit.
• Optimized the QIAseq protocol for low RNA concentrations using RNA extracted from 100 µL plasma.

Main Results:

• Initial RNA extraction kits yielded low miRNA library concentrations (0–1.42 ng/µL).
• Optimizing the QIAseq protocol increased average miRNA library yields to 5.6 ng/µL (max 24.3 ng/µL) across 92 samples.
• The optimized protocol showed no age or gender bias.

Conclusions:

• The optimized QIAseq protocol significantly improved miRNA library yields from low-volume pediatric plasma.
• An 8.7% failure rate was observed, which is rarely reported, but represents an improvement for this sample type.
• The optimized protocol supports further research into pediatric circulating miRNAs.