Optimised lyophilisation-based method for different biomolecule single-extractions from the same rat brain sample:...

Area of Science:

• Neuroscience Research
• Preclinical Biomarker Discovery
• Animal Model Studies

Background:

• Optimizing tissue processing in preclinical studies is crucial for reducing animal usage and enabling integrated, multi-level analyses from single samples.
• Current methods for multiple biomolecule extractions from the same sample present significant limitations.

Purpose of the Study:

• To develop and validate a novel method for optimizing tissue handling and preservation of rat brain samples subjected to ischemic stroke.
• To enable independent RNA and protein single-extractions for subsequent RT-qPCR and Western blot analyses from the same tissue sample.

Main Methods:

• Combined lyophilisation (freeze-drying) of flash-frozen rat brain tissue with mechanical pulverisation and cryopreservation.
• Developed a protocol for optimizing tissue handling and preservation for independent RNA or protein single-extraction methods.
• Utilized subsequent RT-qPCR and Western blot analyses to assess biomolecule integrity and expression.

Main Results:

• Lyophilisation resulted in a 70% tissue weight loss, but yielded comparable RNA and protein quantities to non-lyophilized samples.
• RNA and protein yields were consistent between non-ischemic and ischemic brain samples, regardless of flash freezing (FF) or flash freezing plus lyophilisation (FF+Lyo).
• RNA transcription of reference genes and expression of housekeeping proteins, as well as stroke-regulated gene mRNA, were similar in both FF and FF+Lyo samples.

Conclusions:

• The lyophilisation-based method allows for multiple, representative biomolecule extractions from aliquots of the same rat brain tissue powder.
• This approach is sample-saving, supports the reduction principle in animal research, and facilitates coordinated transcriptome and proteome analyses.
• The method is cost-effective compared to commercial kits and offers higher yields than multiple manual or kit-based extractions, maintaining sample integrity unlike heat-based drying methods.