RNA from stabilized whole blood enables more comprehensive immune gene expression profiling compared to RNA from peripheral blood mononuclear cells.

Fleur Van Der Sijde,Rick Schraauwen,Casper H J Van Eijck,Yunlei Li,Dana A M Mustafa,Willem De Koning,Francesco Bertolini

doi:10.1371/journal.pone.0235413

Abstract

Monitoring changes in the immune profile in blood samples can help identifying changes in tumor biology and therapy responsiveness over time. Immune-related gene expression profiles offer a highly reproducible method to monitor changes of the immune system. However, measuring gene expression profiles in whole blood samples can be complicated because of the high protein and enzyme abundancy that affect the stability and quality of the RNA. Peripheral blood mononuclear cells (PBMCs) are one the most commonly used source for immune cell RNA extraction, though, this method does not reflect all components of the peripheral blood. The aim of this study was to determine the differences in immune-related gene expression between RNA isolated from stabilized whole blood and RNA isolated from PBMCs. Whole blood samples from 12 pancreatic cancer patients were collected before and after chemotherapy (n = 24). Blood samples were collected in both EDTA tubes, and Tempus tubes containing an RNA stabilizer (total n = 48). PBMCs were isolated from EDTA samples using Ficoll and were snap frozen. Subsequently, immune-related gene expression was profiled using the PanCancer Immune Profiling Panel of NanoString technology. Gene expression profiles of PBMCs were compared to that of Tempus tubes using the Advanced Analysis module of nSolver software. Both types of samples provided good quality RNA and gene expression measurements. However, RNA isolated from Tempus tubes resulted in significantly higher gene counts than PBMCs; 107/730 genes were exclusively detected in Tempus samples, while under the detection limit in PBMCs. In addition, 192/730 genes showed significantly higher gene counts in Tempus samples, 157/730 genes showed higher gene counts in PBMCs. Thus, RNA isolated from whole blood stabilizing blood tubes, such as Tempus tubes, enable higher gene counts and more comprehensive measurements of gene expression profiles compared to RNA isolated from PBMCs.

Highlights

In this era of rapidly evolving immune monitoring and immune therapies in many different research fields, longitudinal peripheral blood immune profiling is becoming crucial to monitor the outcome of patients [1]
Peripheral blood is the main route for transportation of immune cells, which thereby provides the opportunity to monitor the activity of the immune system [3, 4]
We compared the abundancy of measured immune-related genes, sensitivity of detection and the range of identified immune cells between RNA isolated from Peripheral blood mononuclear cells (PBMCs) and RNA isolated from whole blood samples collected in Tempus tubes containing a stabilizing reagent

Summary

Introduction

In this era of rapidly evolving immune monitoring and immune therapies in many different research fields, longitudinal peripheral blood immune profiling is becoming crucial to monitor the outcome of patients [1]. Peripheral blood sampling is a low-risk, non-invasive procedure and available at low costs [2]. Peripheral blood is the main route for transportation of immune cells, which thereby provides the opportunity to monitor the activity of the immune system [3, 4]. Monitoring the immune system requires more than profiling subtypes of immune cells. Blood-based gene expression profiles reflect activity of the immune response, including all types of circulating immune cells, their secreted cytokines, chemokines, and neoantigens

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