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Background Optimisation of tissue processing procedures in preclinical studies reduces the number of animals used and allows integrated multilevel study in the same sample. Multiple extraction of different biomolecules from the same sample has several limitations. New method Using brain samples from rats subjected to ischemic stroke, we combined lyophilisation of flash-frozen tissue, mechanical pulverisation and cryopreservation in a method to optimise tissue handling and preservation for independent RNA or protein single-extract methods, and subsequent RT-qPCR or Western blot analyses. Results Lyophilisation resulted in 70% tissue weight loss. RNA (OD260/280∼1.8) and protein yields were similar in non-ischemic and ischemic brain samples, subjected to either flash freezing (FF) or flash freezing followed by lyophilisation (FF + Lyo). RNA transcription of reference genes (Actb and Rn18s), expression of housekeeping proteins (β-actin and α-tubulin), and mRNA overexpression of stroke-regulated genes (Nos2, Mmp9 and Tnfa) was similar in FF and FF + Lyo samples. Comparison with existing method(s) Contrary to high heat stress of baking method in a drying oven, lyophilisation maintains the integrity of dried samples for subsequent extractions and analyses. Sample lyophilisation allows different manual representative extractions/analyses from the same rat, it is much cheaper than using commercial kits, and shows higher yields that multiple manual or kit-based extractions. Conclusions The lyophilisation-based method for different biomolecule single-extractions from tissue powder aliquots, representing the same rat brain sample, is sample saving, contributes to the reduction principle in animal research, and allows coordinated analysis for accurate correlations between the transcriptome and proteome in stroke and other neuroscience research.