Folia Biologica
Journal of Cellular and Molecular Biology, Charles University 

Although next-generation RNA sequencing (RNA-seq) is increasingly incorporated into germline cancer predisposition testing, its diagnostic utility is often limited by low expression of many clinically relevant genes. To improve RNA yield and transcript representation for targeted RNA-seq, we optimized a simple protocol based on short-term lymphocyte culture prepared directly from whole blood collected in Li-heparin tubes. We systematically evaluated biological reproducibility and pre-analytical sample handling variability and demonstrated that whole blood can be stored at 4 °C for up to 5 days prior to lymphocyte cultivation without compromising RNA quality/gene expression. Gene expression was comparable for RNA isolated from K₂EDTA and Tempus tubes, whereas short-term lymphocyte culture resulted in a substantial increase in expression of clinically important genes including BRCA1, BRCA2, RAD51C, RAD51D, PALB2, CHEK2, and multiple Fanconi anaemia genes otherwise low expressed in whole blood. Cultivation for 3–5 days did not significantly affect lymphocyte gene expression, providing flexibility for routine dia­gnostics. The protocol also enables inhibition of nonsense-mediated decay to facilitate analysis of variants causing premature termination. As a proof of principle, we characterized the splicing impact of FANCA c.2602-3C>G variant (located in intron 27) using cultured lymphocytes from its carrier. The variant causes deletion of six nucleotides in the mature transcript (ΔE28p(–6)/r.2602_2607del), resulting in an in-frame deletion (p. Gln869_Phe870del). This spliceogenic effect was reliably detectable only in cultured lymphocytes preferentially expressing the full-length FANCA transcript. Overall, short-term lymphocyte culture re­presents a simple and flexible RNA source that enhances variant interpretation in clinical RNA-seq analyses.

Supplementary materials: Tables S1-S9 (xlsx)

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