Abstract
Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species.
Highlights
Global transcriptomic profiling is a useful tool to identify novel biomarkers for disease and toxicity [1,2,3,4]
A number of studies have demonstrated that transcriptomic changes in peripheral blood can serve as biomarkers of exposure to xenobiotics or as biomarkers for pathological changes occurring in other tissues [5,6,7,8]
Total RNA was isolated from 2.5 mL rat whole blood using the standard PAXgene method or from
Summary
Global transcriptomic profiling is a useful tool to identify novel biomarkers for disease and toxicity [1,2,3,4]. Up-regulation of genes related to hypoxia and down-regulation of genes related to metabolism and cell cycle have been observed in whole blood samples when RNA was not immediately isolated after blood collection [11] Attempts to overcome these hurdles led to the development of a number of new approaches for blood RNA stabilization [13]. One of those is the PAXgene blood RNA system that has been widely used in clinical settings for blood transcriptomic studies. Using blood from rats exposed to lipopolysaccharide (LPS) as a model system, the present study demonstrates that the RNA isolated by the proposed QIAzol-based stabilization and isolation method (QSI) can provide robust transcriptomic profiles comparable to those obtained from RNA isolated with the standard
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