Abstract B019: RNA transcriptome profiling in microsamples of blood

Abstract

Abstract Multi-omic analysis of microsamples of lancet-induced blood drops allows frequent capture and quantitation of numerous metabolites, lipids, cytokines, and proteins. Microsample-based transcriptome profiling would facilitate use of RNA biomarkers in diagnosis and treatment of immunotherapy patients, but a suitable method has not been available. We tested a targeted sequencing protocol for this purpose in human blood microsamples. We tested normal blood collected either by phlebotomy into standard vacutainer tubes or by absorption of 30 uL of blood onto a Mitra device pre-treated with an RNA-stabilization reagent. We tested detection of gene expression changes by incubating anti-coagulated blood for 18 hours with endotoxin followed by isolation of identical volumes of blood using a standard method (Qiagen kit) or from Mitra devices air dried for 24 h to mimic a home-use scenario. RNA was extracted from the microsamplers. After RNA purification, a panel of 274 immune/inflammatory genes was quantified by NGS after PCR using targeted primers following the Cellecta DriverMap protocol, which avoids counting of abundant rRNA and mitochondrial sequences. The targeted sequencing results were normalized to counts per million and used to compare results in standard vs microsamples. We found robust detection of gene expression and correlation using the two methods in both unstimulated (r = 0.94) and endotoxin-stimulated blood. The differentially expressed genes (DEGs) identified in both standard and microsample methods showed high overlap with DEGs reported in public datasets in similar experiments. We conclude that RNA transcriptome profiling using targeted sequencing allows sensitive detection of gene expression levels in normal and activated blood samples. Because microsamples can be air-dried and mailed in for analysis, this approach has great promise for simple and repeated monitoring of RNA biomarkers in immunotherapy. Citation Format: Alex Chenchik, Lester Kobzik, Mikhail Makhanov, Paul Diehl. RNA transcriptome profiling in microsamples of blood [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B019.