Yao Yao, Wang Fen
Department of Gastroenterology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2020 Aug 28;45(8):909-915. doi: 10.11817/j.issn.1672-7347.2020.190083.
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.
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.
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 >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 (<0.05), while the absorbance value of RNA was increased in the 9th and 12th months (both >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 , and was decreased significantly, while the abundance of was increased significantly. At the family level, the abundance of , and was lower than that in the fresh sample, and the abundance of 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 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.
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.
比较不同保存方法在不同时间点对生物大分子和微生物菌群的影响，了解不同冷冻保存方法、保存时间与样本稳定性之间的关系。
从中南大学湘雅三医院接受根治性胃切除术的患者中采集组织样本。从志愿者中获取粪便样本。组织样本分别储存在-80℃冰箱（-80℃冰箱组）和液氮罐（液氮罐组）中。根据保存方法，粪便样本分为-80℃冰箱组、液氮罐组、-80℃冰箱+无水乙醇组、液氮罐+无水乙醇组和室温+无水乙醇组。分别在0个月（新鲜样本）、3个月、6个月、9个月和12个月检测并分析相关指标，以评估不同保存方法下样本的稳定性。
与新鲜样本相比，液氮罐组样本在储存12个月后蛋白质浓度和RNA吸光度值均无显著变化（均>0.05）；但-80℃冰箱组组织样本在储存12个月后蛋白质浓度降低（<0.05），而RNA吸光度值在第9个月和第12个月升高（均>0.05）。对于粪便样本的微生物菌群，在门水平上，室温+无水乙醇组菌群随时间变化显著， 、 和 的丰度显著降低，而 的丰度显著增加。在科水平上， 、 和 的丰度低于新鲜样本，而 的丰度高于新鲜样本。在4个冷冻保存组（-80℃冰箱组、液氮罐组、-80℃冰箱+无水乙醇组、液氮罐+无水乙醇组）中， 的丰度高于新鲜样本。Alpha和Beta多样性分析表明，室温组样本的物种多样性、丰富度和菌群结构与新鲜样本及其他实验组不同。
液氮罐保存的组织样本中蛋白质浓度和RNA纯度相对稳定；-80℃冰箱保存的组织样本在半年内蛋白质浓度和RNA纯度也相对稳定；-80℃冰箱和液氮罐冷冻保存均可维持粪便标本的微生物菌群稳定性；室温+无水乙醇不适合长期保存粪便样本。
