Rescuing RNAs from heparin-contaminated blood samples for RNA sequencing

Pre-analytical variations, particularly apoptotic stress responses in blood samples, remain a critical challenge in transcriptomic data harmonization. A major source of this noise is the interference caused by anticoagulants, specifically heparin, which inhibits downstream enzymatic reactions and induces ex vivo stress artifacts that confound gene expression profiles Unlike traditional immediate lysis methods, our approach introduces lithium chloride (LiCl) prior to sample storage to exert a protective effect that mitigates stress-induced cellular damage and reduces apoptosis. Furthermore, LiCl is applied again prior to the RNA precipitation step to serve as a decontamination agent. This LiCl-based protocol is particularly effective for rescuing archived blood samples contaminated with heparin and stored under long-term frozen conditions (-80 °C for up to 180 days). Key results demonstrate that LiCl treatment significantly restores RNA integrity (P < 0.01), achieving an average RIN value > 7.0. Notably, we observed that adding LiCl during the storage of hypoxia-stressed whole blood samples significantly reduced cell apoptosis (P < 0.001). Consequently, little inter-sample variability was observed, and the transcriptomic profiles of rescued samples show high concordance with those from EDTA-anticoagulated controls, enabling high-quality sequencing without the need for complex computational normalization. Thus, this LiCl-based method offers a robust, cost-effective, and environmentally friendly alternative for high-fidelity blood transcriptomics, especially valuable for precious clinical cohorts where re-sampling is not feasible. However, due to LiCl's selective precipitation of larger RNA fragments (>200 nt), this protocol is not suitable for analyses targeting small RNAs such as microRNAs.