FastEV & mRNA sequencing

FastEV performs well in mRNA sequencing

RNA form FastEV was subjected to next generation messenger RNA sequencing (mRNAseq) at University of Helsinki, FIMM. The FastEV conditions gave the highest numbers of reads, and particularly high exonic and low intronic read distributions compared to EV and plasma controls. The number of detected genes was generally at similar level, close to 20 000 genes, from all samples.

Figure: Number of reads obtained from the FastEV samples in mRNAseq. The read counts from most FastEV conditions were higher than from EV preparation (PEG, UC) or plasma controls. Data is from one technical replicate per sample, except in case of three FastEV conditions (n=3) and controls (n=2). Error bars show SD for replicates. PEG, polyethylene glycol, UC, ultracentrifugation.

Figure: Number of detected genes. The number of detected genes at different cut-offs (1 and 5 counts) indicated that all samples contained around 20 000 transcripts. FastEV conditions yielded gene numbers that were mostly intermediate between those from EV (PEG, UC) and plasma controls. Data is from one technical replicate per sample, except in case of three FastEV conditions 1-3 (n=3) and controls (n=2). Error bars show SD. PEG, polyethylene glycol, UC, ultracentrifugation.

Figure: Distribution of reads over genomic features. ​FastEV conditions provided the highest numbers of reads from exons and lower intronic or intergenic reads than EV preparation controls (PEG, UC). Data is from one technical replicate per sample, except in case of three FastEV conditions 1-3 (n=3) and controls (n=2). CDS, coding sequence; PEG, polyethylene glycol; TES, transcriptional end site, TSS, transcriptional start site; UC, ultracentrifugation.

Methods: Analysis of FastEV conditions with mRNAseq​

FastEV conditions and control EV enrichment methods (UC, PEG) were used for a pooled plasma sample. Then, total RNAs were extracted from the samlpes for mRNA sequencing. For details, see basics/RNA. Messenger RNAs were sequenced using mRNAseq protocol based on poly A-priming, a method well established for EV-RNAs (Barreiro et al., 2020) and plasma RNAs (Nevalainen et al., 2021). Study included one technical replicate per sample, except in case of three specific FastEV conditions 1-3 (n=3) and all controls (n=2). Raw read counts were filtered to exclude genes with very low counts using R package edgeR. Reads were normalised using variance-stabilizing transformation of R package DESeq2. For PCA, top 500 variable genes were used. For heatmap, top 150 most variable genes were selected and Log2 fold changes calculated between different conditions and plasma. Both columns and the rows were hierarchically clustered using Euclidean distances and complete linkage method.

FastEV provides differential RNA contents for biomarker discovery

Principal component analysis (PCA) of mRNAs showed clear differences between FastEV conditions and controls. Thus, FastEV demonstrated its capacity to bring different RNA contents for the biomarker discovery.

Figure: Principal component analysis shows differences and similarities between FastEV conditions and controls. PCA plot was generated using the top 500 most variable genes. FastEV conditions separated from the controls (UC, PEG and plasma) and formed few distinct clusters separated by both PC1 and PC2. Data is from one technical replicate per sample, except in case of three FastEV conditions 1-3 (n=3) and controls (n=2). PC, principal component, PCA, principal component analysis, PEG, polyethylene glycol, UC, ultracentrifugation.

FastEV enriches and depletes RNAs from plasma

FastEV conditions were capable of enriching and depleting unique sets of transcripts from plasma.

Figure: Heatmap of fold changes vs plasma of top 150 differential mRNAs. FastEV conditions enriched or depleted specific sets of RNAs from plasma, the heatmap shows log2 fold change compared to plasma. While the expression patterns clearly varied, some conditions resembled each other and clustered together in hierarchical clustering. Yellow color indicates level most similar to plasma, red indicates enrichment and blue depletion relative to plasma. Data is from one technical replicate per sample, except in case of three FastEV conditions 1-3 (n=3) and controls (n=2). PC, principal component, PEG, polyethylene glycol, UC, ultracentrifugation.​