A Novel Technology for Multiplex Gene Expression Analysis Directly from Whole Blood Samples Stabilized at Ambient Temperature Using an RNA-Stabilizing Buffer

Abstract

We describe a novel method, based on target-dependent chemical ligation of probes, which simplifies the multiplexed quantitation of gene expression from blood samples by eliminating the RNA purification step. Gene expression from seven genes was evaluated over a range of sample inputs (16.7 to 0.25 μL of whole blood in serial dilutions) from three healthy donors. Mean CVs were ≤11% for five technical replicates for whole blood inputs ≥2.1 μL. The method showed a limit of detection of 300 copies of RNA by using titration of in vitro transcripts for four genes. Gene expression measured on stabilized blood samples was highly correlated (Spearman rank correlation method, ρ = 0.80) to gene expression results obtained with RNA isolated from matched samples (three donors, five technical replicates). Gene expression changes determined with seven radiation-responsive genes on six healthy donor blood samples before and after ex vivo irradiation were highly correlated (ρ = 0.93) to those measured with a TaqMan quantitative real-time RT-PCR assay on RNA purified from matched samples. Thus, this method is reproducible, sensitive, and correlated to quantitative real-time RT-PCR and may be used to streamline the multiplex gene expression analysis of large numbers of stabilized blood samples without RNA purification.

A Meta-Analysis of the Performance of a Blood-Based Exposure Response Gene Signature Across Clinical Studies on the Tobacco Heating System 2.2 (THS 2.2)

“…The blood-based gene signature could also be transferred to an easy-to-use and economic kit for smaller blood volumes to facilitate clinical use. Such kits and technologies already exist to evaluate radiation exposure (Lucas et al, 2014; Kim et al, 2015). These kits are simple to use at different medical sites and even by subjects in their own homes.…”

Type 1 interferon status in systemic lupus erythematosus: a longitudinal analysis

“…IFN status was measured by DxTerity Diagnostics (Rancho Dominguez, California, USA), using the DxTerity Modular Immune Profile test, a commercially available chemical ligation-dependent probe amplification and gene expression test with relative quantitative analysis by capillary electrophoresis. Sample testing and analysis was performed directly on PAXgene RNA Stabilized Blood as described by Kim et al 20 with minor modifications to the protocol. The DxTerity 4-gene Type 1 Interferon (IFN-1) test measures the RNA expression levels of four ISGs (HERC5, IFI27, IFIT1 and RSAD2) to the expression levels of three housekeeping normaliser genes (ACTB, GAPDH and TFRC).…”

Deucravacitinib, a Tyrosine Kinase 2 Inhibitor, in Systemic Lupus Erythematosus: A Phase II, Randomized, Double‐Blind, Placebo‐Controlled Trial

“…Prespecified exploratory end points analyzed at week 48 included the percent of patients achieving Joint Count‐50 (a decrease in active joint count of ≥50% from baseline in patients with ≥6 active joints at baseline) ( 28 ); time to SRI‐4 response; and change from baseline in mean complement (C3, C4) and anti–double‐stranded DNA levels. Change from baseline in type I IFN gene signature in patients’ whole blood was determined by a central laboratory (DxTerity) using an analytically validated 5‐gene ( MX1, HERC5, IFIT1, RSAD2, EIF2AK2 ) chemical ligation–dependent probe amplification–based test ( 29 ).…”