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

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.

A Toolkit for Profiling the Immune Landscape of Pediatric Central Nervous System Malignancies

“…With bulk RNA-seq, evaluating the expression of immune-related genes and enrichment of immune-related pathways can reveal the inflammatory state of the TIME ( 29 , 90 ). A possible, and promising, solution is to perform RNA-seq on immune cells found in blood or CSF, which could be correlated to the TIME ( 93 , 156 ). Although, to our knowledge, no such experiments have been performed to monitor pediatric CNS malignancies yet, studies on other cancers have shown the potential of using these more readily accessible biomaterials to monitor the anti-tumor immune response during therapy.…”

“…Although, to our knowledge, no such experiments have been performed to monitor pediatric CNS malignancies yet, studies on other cancers have shown the potential of using these more readily accessible biomaterials to monitor the anti-tumor immune response during therapy. For example, circulating miRNA levels were found to be correlated to the absolute neutrophil count in pancreatic cancer ( 93 ). Moreover, comparing immune signatures of patients who respond to those who do not respond to a particular type of therapy can eventually guide therapy decision ( 157 ).…”

“…In another study, macrophages and microglia from adult IDH -wt and IDH -mut glioma showed a disease-specific enrichment of inflammatory pathways: IDH -wt macrophages were enriched in gene sets associated with antigen presentation, and MHC I and II presentation ( 57 ). Mononuclear immune cells can also be isolated from the blood or CSF for transcriptomic profiling to define genes and gene sets implicated in various immune cells ( 93 ).…”

Comparative Analysis of the Antitumor Immune Profiles of Paired Radiotherapy-naive and Radiotherapy-treated Cervical Cancer Tissues

“…Expression of immune response-related genes was analyzed using an nCounter Analysis System with a PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) (8). This panel consists of 730 immune response-related genes, covering 24 different immune cell types, and both the adaptive and innate immune responses (14). Analysis was performed as reported previously (9,(14)(15)(16)(17).…”

Distinct CholinomiR Blood Cell Signature as a Potential Modulator of the Cholinergic System in Women with Fibromyalgia Syndrome

“…Blood sample processing and isolation of PBMC prior to analysis affects gene regulation within hours and may affect miR transcription even more rapidly [94][95][96]. In contrast, our approach allowed us to create a direct snap shot of the transcriptomic state, since all cells were immediately lysed and total RNA was stabilized [97]. However, several limitations must be addressed.…”