Lexogen is the Gold Sponsor of the online RNA Society Meeting 2020

Steve W. Cole¹

¹ Department of Psychiatry & Biobehavioral Sciences and Department of Medicine
UCLA School of Medicine
Norman Cousins Center for Psychoneuroimmunology
USC/UCLA Center of Biodemography and Population Health
UCLA Social Genomics Core Laboratory

Genome-wide RNA sequencing of whole blood samples has emerged as a powerful tool for analyzing biological mechanisms underlying social and environmental gradients in health, but utilization in population-based field studies has been hampered by logistical constraints, regulatory strictures, and costs associated with venipuncture blood sampling. Dried blood spots (DBS) provide a minimally invasive, low-cost alternative to venipuncture, that can be readily deployed to collect whole blood samples in field settings without the need for phlebotomy (and potentially by study participant self-collection with sample return by mail).  DBS sampling thus enables the collection of blood biomarker data from research settings where other forms of blood sampling are impossible, as well as analysis of archival clinical samples (e.g., neonate heelstick DBS collected for disease screening).  Surprisingly, desiccation of DBS stabilizes RNA over time by reducing hydrolysis and quickly inactivating RNAses.  However, sequencing of DBS-derived RNA samples is complicated by limited RNA mass available in DBS (~10 ng, < 10% of typical venipuncture RNA) and mild degradation incurred during desiccation and storage (RIN ~5-8).  This talk will describe the application of Lexogen’s QuantSeq 3’ FWD mRNA-seq counting assay to conduct high-quality genome-wide mRNA profiling in DBS samples.   Compared to standard cDNA library preparation systems, QuantSeq is substantially more sensitive (a PCR step amplifies limited input mRNA), more robust to partial RNA degradation (focused sequencing of only the 3’ portion of mRNA misses most strand breaks), more efficient (maximizing the number of transcripts that can be sequenced in a given lane volume by focused sequencing of the 3’ portion of poly-A transcripts), and more cost-effective (<1/3 cost per transcript assayed).  Due to the limited input RNA mass, QuantSeq assay of DBS samples inevitably yields greater noise than parallel analyses of high-mass RNA samples, and this talk will also describe the development of methods to mitigate those effects through specialized study designs (particularly enhanced sample size and repeated measurement), specialized post-run QC screening, and specialized statistical and bioinformatics analyses of transcript abundance data (e.g., focusing on pathways and other gene set measures to smooth over individual transcript-level noise).   The Lexogen Sequencing Services team has experience conducting the specialized cDNA preps needed for DBS samples, and the NIH-funded UCLA Social Genomics Core Laboratory can provide additional consulting support in specialized study design and data analyses for DBS.  This presentation will illustrate the substantive value of DBS RNA sequencing in a study of inflammatory signaling and pregnancy outcomes in archival samples and a randomized controlled field study of behavioral interventions.  DBS should not supplant standard methods when venipuncture is feasible, but it does open up the potential for genome-wide transcriptional profiling in a wide range of archival and field studies where transcriptomic analyses were previously infeasible.