FastEV & mRNA comparisons

Comparison of three FastEV conditions reveals dominant upregulation of genes in condition 3, but also significant unique changes per condition

Differential gene expression analysis of three FastEV conditions revealed a dominant upregulation of approximately 5% of the detected transcriptome in condition 3 vs 1 or 2. Conditions 1 and 2 differed less from each other. Looking at the identities of the differential transcripts, >60% were the same in conditions 1 and 2, while >20% were uniquely differential in either condition.

Methods: Comparison of mRNAseq results of three FastEV conditions ​

Three FastEV conditions (1-3) were used to isolate replicate samples (n=3) from pooled plasma of healthy controls. Results were compared in terms of differential gene expression, where p-value of <0.001 or FDR <0.01 were considered significant. For the analysis of Gene ontology terms (GO terms), p adj <0.05 was considered significant. ​

Table: The number of differentially expressed genes between FastEV conditions 1-3. ​FDR, false discovery rate, FC, fold change.

Figure: Venn comparison of the differentially expressed mRNAs. ​Changes of over 1000 genes were shared between comparisons of condition 3 vs 1 and 3 vs 2. However, both comparisons indicated also >300 uniquely differential mRNAs. Five mRNAs were expressed differentially between all three conditions. Differential genes with FDR<0.01 were included in the analysis.

Figure: Five mRNAs expressed at different levels in three FastEV conditions. The five shared deregulated mRNAs were derived from the Venn comparison. Bar graph presents fold changes of individual comparisons: the expression of all genes was highest in condition 3 followed by 1 and lowest in condition 2. Differential genes with FDR<0.01 were included in the analysis. FC, fold change. IL23R, Interleukin 23 Receptor; NAV2, Neuron Navigator 2; MAGI1, Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 1; CHPF, Chondroitin Glucuronyltransferase 2; STARD9, StAR Related Lipid Transfer Domain Containing 9.

Gene ontology analysis of three FastEV conditions – condition 3 enriches central nervous system –linked ontologies

FastEV condition 3 enriched many Gene ontology terms that are hot topics in studies of e.g. central nervous system diseases. The analysis was conducted on differentially expressed mRNAs between conditions 1-3.

Figures: Significant Gene ontologies between FastEV conditions 1-3. Bubble charts present the most significant (p adj. <0.05) GO terms linked to differential transcripts. Comparisons between condition 3 vs either 1 or 2 highlighted particulary neuronal, transporter, membrane and extracellular processes, components and functions. The sizes of the foreground bubbles indicate the number of significant mRNAs, whereas the sizes of the light blue background bubbles indicate the number of annotated mRNAs of the GO Term. GO, gene ontology.

Three FastEV conditions enrich unique or shared sets of transcripts from plasma – depletion patterns are highly unique

Differentially expressed genes in comparison to plasma revealed unique sets of up- and downregulated genes in samples. The FastEV conditions and EV controls shared a set of common upregulated genes, while there were zero common downregulated genes.

Methods: Analysis of mRNAseq results of FastEV vs plasma​

Pooled plasma from healthy controls was subjected to isolation with FastEV conditions 1-3 (n=3 technical replicates each) and EV control methods (PEG and UC, both n=2). Results were compared to data from replicate samples of the plasma pool (n=2). Differential gene expression was analyzed using Deseq2 and results with p adj <0.05 and fold change >2 considered significant. Venn comparisons were used to derive numbers and lists of uniquely or commonly up/downregulated genes (vs plasma), which were further analyzed with Enrichr tools (Chen et al., 2013; Kuleshov et al., 2016) for GO terms (Biological processes 2018, Molecular functions 2018, Cellular compartments 2018), Jensen compartments and Reactome 2016. Items with p adj <0.05 were considered significant.​

Figures: Venn comparison of the up- and downregulated genes in three FastEV conditions or EV isolation controls versus plasma. Conditions 1 and 3 and PEG control showed the highest numbers of uniquely upregulated genes compared to plasma. All three FastEV conditions (1-3) and EV controls (UC, PEG) shared upregulation of 58 genes, whereas they did not share any downregulated genes. Condition 1 and both EV controls had a high number of unique downregulated genes relative to plasma. Venns present differential genes with FC >2 and p adj <0.05.

FastEV-enriched gene sets regulate specific pathways, functions and compartments

FastEV conditions enriched and depleted specific pathways or components from plasma. For example, condition 1 enriched platelet vesicle –related, condition 2 transcription-related and condition 3 mitochondria -related items.

Table: The main pathways, functions or compartments regulated by the unique differentially expressed genes between three FastEV conditions or EV controls vs plasma. Uniquely differential gene lists were obtained from Venns and analyzed by Enrichr tool for GO terms (Biological processes, Molecular functions, Cellular compartments), Jensen compartments and Reactome. Key words describing the most significant findings are presented in the table. GO, gene ontology.

Platelet vesicles

Transcription

Mitochondria

Figures: Most significant terms from Gene ontology and Jensen compartments analysis. Uniquely upregulated genes vs plasma were obtained from Venns and analyzed by Enrichr tool for GO terms (Biological processes in green, Molecular functions in gray, Cellular compartments in blue) and Jensen compartments (in pink). The terms with p adj <0.05 are shown. No terms reached this significance level in EV controls obtained by ultracentrifugation and polyethylene glycol precipitation. GO, Gene ontology; JC, Jensen compartments.