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Effects of different preservation methods on biomacromolecules and microbial flora.

OBJECTIVES
To compare the effects of different preservation methods on biomacromolecules and microbial flora at different time points, and to understand the relationship between different cryopreservation methods, storage time and sample stability.


METHODS
Tissue samples were taken from patients who underwent radical gastrectomy in the Third Xiangya Hospital, Central South University. Stool samples were obtained from volunteers. The tissue samples were stored in a refrigerator at -80 ℃ (-80 ℃ refrigerator group) and a liquid nitrogen tank (liquid nitrogen tank group), respectively. According to the preservation method, the stool samples were divided into a -80 ℃ refrigerator group, a liquid nitrogen tank group, a -80 ℃ refrigerator+ethanol absolute group, a liquid nitrogen tank+ethanol absolute group, and a room temperature+ethanol absolute group. Relevant indexes were examined and analyzed at the 0 month (fresh sample), 3th month, 6th month, 9th month and 12th month respectively to evaluate the sample stability with different preservation methods.


RESULTS
Compared with the fresh sample, there was no significant changes in the protein concentration and the absorbance value of RNA in the samples in the liquid nitrogen tank group after 12 months of storage (both P>0.05); but the protein concentration of the tissue samples was decreased in the samples of the -80 ℃ refrigerator group after 12 months of storage (P<0.05), while the absorbance value of RNA was increased in the 9th and 12th months (both P>0.05). For the microbial flora of feces samples, at the phylum level, the flora changed significantly with time in the room temperature+ethanol absolute group, and the abundance of bacteroidetes, clostridium and proteobacteria was decreased significantly, while the abundance of firmicutes was increased significantly. At the family level, the abundance of bacteroidaceae, trichospirillaceae and veroniaceae was lower than that in the fresh sample, and the abundance of rumenbacteriaceae was higher than that in the fresh sample. In the 4 cryopreserved groups (-80 ℃ refrigerator group, liquid nitrogen tank group, -80 ℃ refrigerator+ethanol absolute group, liquid nitrogen tank+ethanol absolute group), the abundance of enterobacteriaceae was higher than that in the fresh sample. The analysis of Alpha and Beta diversity indicated that the species diversity, richness and flora structure of samples in the room temperature group were different from those in the fresh sample and other experimental groups.


CONCLUSIONS
The protein concentration and RNA purity in the tissue samples preserved by liquid nitrogen tank are relatively stable; the protein concentration and RNA purity of the tissue samples which preserved in the -80 ℃ refrigerator are also relatively stable within half a year; the -80 ℃ refrigerator and liquid nitrogen tank cryopreservation can both maintain the microbial flora stability of the fecal specimen; the room temperature+ethanol absolute is not suitable for long-term preservation of stool samples.