The effect of cell isolation methods on the human transcriptome profiling and microbial transcripts of peripheral blood

Abstract

The expression of human and microbial genes serves as biomarkers for disease and health. Blood RNA is an important biological resource for precision medicine and translational medicine. However, few studies have assessed the human transcriptome profiles and microbial communities composition and diversity of peripheral blood from different cell isolation methods, which could affect the reproducibility of researches. We collected peripheral blood from three healthy donors and processed it immediately. We used RNA sequencing to investigate the effect of three leukocyte isolation methods including buffy coat (BC) extraction, red blood cell (RBC) lysis and peripheral blood mononuclear cell (PBMC) isolation with the comparison with whole blood (WB), through analyzing the sensitivity of gene detection, the whole transcriptome profiling and microbial composition and diversity. Our data showed that BC extraction with high globin mRNA mapping rate had similar transcriptome profiles with WB, while RBC lysis and PBMC isolation depleted RBCs effectively. With the efficient depletion of RBC and distinct compositions of leukocyte subsets, RNA-seq of RBC lysis and PBMC isolation uniquely detected genes from specific cell types, like granulocytes and NK cells. In addition, we observed that the microbial composition and diversity were more affected by individuals than isolation methods. Our results showed that blood cell isolations could largely influence the sensitivity of detection of human genes and transcriptome profile.

Plasma-free samples for transcriptomic analysis: a potential alternative to whole blood samples

“…Given these concerns, our study aimed to assess the viability of employing either PFB or SFB samples as substitute RNA sources in transcriptomic analysis. Previous research have demonstrated the influence of pre-analytical factors on RNA quality and gene expression (Debey-Pascher et al 2011;Mastrokolias et al 2012;Dvinge et al 2014;Shin et al 2014;Zhao et al 2014;Huang et al 2017;Reust et al 2018;Shen et al 2018;Donohue et al 2019;Gautam et al 2019;He et al 2019b;Harrington et al 2020;Xing et al 2021;Chebbo et al 2022;Husseini et al 2022). However, a comprehensive understanding of the similarities and differences among WB, PFB, and SFB samples in transcriptomic research remains elusive, making it crucial to evaluate their suitability for various applications using reliable metrics.…”

Plasma-Free Blood as a Potential Alternative to Whole Blood for Transcriptomic Analysis

“…Given these concerns, our study aimed to assess the suitability of employing either PFB or SFB samples as alternative RNA sources in transcriptomic analysis. Previous researches have demonstrated the influence of pre-analytical factors on RNA quality and gene expression (Debey-Pascher et al 2011 ; Mastrokolias et al 2012 ; Dvinge et al 2014 ; Shin et al 2014 ; Zhao et al 2014 ; Huang et al 2017 ; Reust et al 2018 ; Shen et al 2018 ; Donohue et al 2019 ; Gautam et al 2019 ; He et al 2019a ; Harrington et al 2020 ; Xing et al 2021 ; Chebbo et al 2022 ; Husseini et al 2022 ). However, a comprehensive understanding of the similarities and differences among WB, PFB, and SFB samples in transcriptomic profiles remains elusive, making it crucial to evaluate their suitability for various applications using reliable performance metrics.…”