FAA Office of Aerospace Medicine
Civil Aerospace Medical Institute

Report No: DOT/FAA/AM-23/01

Title and Subtitle:An Evaluation of the Downstream Effects of Purification Methods on RNA-Seq Differential Expression

Report Date:January 2023

Authors:Munster SK, Uyhelji HA, Nicholson SJ

Abstract:Ribonucleic acid sequencing (RNA-Seq) is a valuable and commonly used technique to quantify the number of individual RNA transcripts within a sample. RNA-Seq typically requires a small amount of pure and concentrated RNA, which can necessitate additional concentration or purification of previously isolated RNA samples. Magnetic beads and silica-based columns are often used to concentrate and/or purify RNA samples, but little is known about how these techniques influence downstream analyses. In this study, we collected blood from volunteer human subjects and pooled those samples during RNA extraction to minimize variance due to input material. We then purified aliquots of that sample pool to evaluate how sample purification and concentration influenced gene expression observations. Extracted RNA was sequenced, and the resulting RNA-Seq files were evaluated to determine the degree of differential expression between methods. Differential expression was detected in roughly half of the comparisons made and appeared attributable at least partly to differences in sample concentration and purification techniques.

Key Words:RNA-Seq, sample purification, differential expression

No. of Pages: 34