Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques

Abstract

Lyophilization is a cost-effective method for biological specimen preservation but detailed tissue-specific reference protocols are still lacking. Moreover, data are limited on the long-term stability of proteins and nucleic acids in lyophilized samples.Here, we offer lyophilization protocols for various rat and mouse tissues (kidney, heart, liver, lung, aorta, and skin) coupled with technical hints for optimal sample preparation. We demonstrate that lyophilized samples stored at 4 °C for 20 months can yield protein and RNA of similar quantity and quality to −80 °C storage, while phosphorylated proteins are preserved as well. Freeze-dried and subsequently pulverized samples can provide more consistent, more reliable data especially when investigating focal injuries, such as fibrosis. We developed a protocol for the concentration of biological solutions and achieved 20-times concentration in human peritoneal dialysis effluent solution which enables the previously unattainable detection of proteins in these samples. We established a method for water removal as well as accurate water content measurement of fecal samples, which can be valuable for gut metabolome analysis.Taken together, lyophilization is a valuable tool for the preservation of biological samples with many advantages. We aim to draw attention to the wide range of possibilities offered by freeze drying in pre-clinical or basic research.

Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines

“…While we have established a reliable freezing protocol for long-term storage of LNP-formulated replicon vaccines, lyophilization to keep vaccines in a dry powder form is another clinically-relevant storage strategy. Dry solids simplify aseptic handling procedures, decrease sample weight and volume for easier shipping, improve stability, and allow easy dose adjustments by dissolution at desired concentrations [ [52] , [53] , [54] ]. However, freeze drying has proven to be challenging for LNP-RNA formulations [ 29 , 35 , 39 ].…”

Dissolved organic metabolite extraction from high‐salt media

“…In this process, the solvent sublimates from the sample, going directly from the solid to the gas phase 25 . Lyophilization has been shown to provide consistent results with strong reproducibility and minimal degradation of metabolites 29 . We transitioned to lyophilization in the middle of the DMSO‐d 6 stage (in 3.7) of method development.…”

Properties of Human Gastric Lipase Produced by Plant Roots

“…There was no loss of extractable lipase activity in dehydrated roots over a period of 27 months. Although freeze-drying is a well-established method of tissue or protein preservation, long-term studies are lacking [ 25 ]. In order to maintain the activity of proteins upon freeze-drying, a protecting agent must often be added to them [ 26 ].…”