Various Preclinical Models, One Blood Transcriptomics Workflow for Translational Research

The Importance of Blood Samples in Biological ResearchImportance of Blood Sample

Blood is known to be an important tissue type for research. With simple procedures like venipuncture, blood samples are relatively easy to collect and less invasive compared to the collection of other tissues. As it circulates throughout the body, blood carries various cell types including immune cells, thus providing rich information about the immune system. Researchers can apply RNA-Seq for the analysis of the blood transcriptome and gain insights into an individual’s physiological and pathological state. This powerful technique further enables various applications such as identifying gene or transcript markers for disease diagnosis and therapeutics,¹ as well as elucidating the mechanisms underlying different vaccination responses.²

The Effort for a Streamlined, Multi-Species Blood Transcriptome Analysis WorkflowMulti-Species Transcriptomics Workflow

Currently, a lack of standardized blood transcriptomics workflow for multiple preclinical models makes it a challenge to integrate RNA-Seq into routine practice. To address this, a research team in France took the initiative and successfully set up a single RNA-Seq workflow compatible with four different species: human, mouse, rabbit, and non-human primate (NHP, Macaca fascicularis).³

The mouse, the rabbit, and NHPs are widely used as animal models driving scientific advancements in preclinical research.⁴ The mouse is perhaps the most used model in human disease research with reduced maintenance costs, ease of breeding, and availability of various strains.^5,6 The rabbit has also played a crucial role in translational research, including contributing to the development of the rabies vaccine around 150 years ago.⁶ Furthermore, non-human primates share the closest genetic proximity to humans phylogenetically, and are valued in a widespread applications and research.⁴ The selection of these three animal models thus reflect their well-established importance in preclinical research.

In the established workflow, the authors emphasized the importance of appropriate blood sample preservation and RNA extraction to ensure its practicality for both large and small animal models: they recommended preserving whole blood samples with stabilizing reagents upon collection, and implemented an RNA extraction protocol that accommodates a large range of blood volumes. To address DNA contamination, the team incorporated DNase I treatment during RNA cleanup with RNA clean & concentrator. For library preparation the researchers utilized the Zymo-Seq RiboFree Total RNA Library Kit. The kit’s universal rRNA depletion capability allowed them to use a single library prep procedure across all four species, simplifying wet-lab practices.

Following Illumina sequencing and transcriptomic data analysis using a custom pipeline, the researchers confirmed that efficient rRNA depletion was achieved across all four species (i.e., <10% of reads were identified as rRNA), and that globin mRNA depletion was effective in most samples (i.e., <10% of reads). The workflow also produced low variability in the number of protein coding genes detected, regardless of the species and initial amount of extracted blood. Leveraging the RiboFree total RNA-Seq approach, the workflow supports the analysis of mRNAs with poly-A tails, mRNAs without poly-A tails, and other types of transcripts. Additionally, the workflow is compatible with degraded RNA inputs, making it versatile for diverse sample qualities.

The Future of Blood RNA-Seq in Preclinical ResearchFuture of Blood RNA-Seq in Research

The end-to-end blood RNA-Seq workflow established by this group of researchers provides a valuable framework to the scientific community to improve standardization and enable inter-species data comparisons. Its adaptability also allows the workflow to be transferred to other species, further enhancing the ability to identify potential biomarkers for monitoring safety and efficacy of drugs and vaccines. Additionally, this approach helps to streamline and bridge clinical and translational research; now, researchers can use a single RNA-Seq sample preparation workflow to study a variety of model organisms in translational studies, including both preclinical and clinical research settings.