Plasma-free samples for transcriptomic analysis: a potential alternative to whole blood samples

RNA sequencing (RNAseq) technology has become increasingly important in precision medicine and clinical diagnostics and emerged as a powerful tool for identifying protein-coding genes, performing differential gene analysis, and inferring immune cell composition. Human peripheral blood samples are widely used for RNAseq, providing valuable insights into individual biomolecular information. Blood samples can be classified as whole blood (WB), plasma, serum, and remaining sediment samples, including plasma-free blood (PFB) and serum-free blood (SFB) samples. However, the feasibility of using PFB and SFB samples for transcriptome analysis remains unclear. In this study, we aimed to assess the viability of employing PFB or SFB samples as substitute RNA sources in transcriptomic analysis and performed a comparative analysis of WB, PFB, and SFB samples for different applications. Our results revealed that PFB samples exhibit greater similarity to WB samples in terms of protein-coding gene expression patterns, differential expression gene profiling, and immunological characterizations, suggesting that PFB can be a viable alternative for transcriptomic analysis. This contributes to the optimization of blood sample utilization and the advancement of precision medicine research. ### Competing Interest Statement The authors have declared no competing interest. * RNAseq : RNA sequencing WB : Whole blood PFB : Plasma-free blood SFB : Serum-free blood PBMCs : Peripheral blood mononuclear cells FPKM : Fragments Per Kilobase of exon model per Million mapped fragments TPM : Transcripts Per kilobase of exon model per Million mapped reads PVCA : Principal variance component analysis DEGs : Differential expression genes CV : Coefficient of variation PCA : Principal component analysis HCA : Hierarchical clustering analysis RPS4Y1 : Ribosomal Protein S4 Y-Linked 1 DDX3Y : DEAD-Box Helicase 3 Y-Linked EIF1AY : Eukaryotic Translation Initiation Factor 1A Y-Linked KDM5D : Lysine Demethylase 5D TXLNGY : Taxilin Gamma Pseudogene, Y-Linked XIST : X Inactive Specific Transcript TSIX : TSIX Transcript, XIST Antisense RNA SD : Standard deviation PCC : Pearson correlation coefficient GO : Gene Ontology KEGG : Kyoto Encyclopedia of Genes and Genomes