高寒区牛内脏组织的采集及其RNA的贮存

Sampling of visceral tissues from Bovines in the Qinghai-Tibet Plateau and techniques of their RNA preservation

牦牛所处环境及生物习性明显不同于普通牛,尤其组织样品获取不易,如何将来之不易的样品长时间高效保存意义重大。为此,本研究以天祝白牦牛及本地黄牛为对象,采集胃肠道、肝脏及肾脏样品,Trizol Kit提取RNA,运用食品药品监督管理局(Food and Drug Administration,FDA)规则及Gel-pro软件分析-80℃保存1个月和23个月的RNA浓度及降解程度,研究RNA的保存与质量检测技术。结果表明,牦牛所有胃组织样品RNA放置23个月后浓度均增加,而黄牛的瘤胃及瓣胃却相反;两个基因型牛种空肠及牦牛回肠组织RNA相对稳定;牦牛和黄牛大肠RNA在保存23个月后浓度存在显著差异(P<0.05)。牦牛及黄牛胃部及肝脏和肾脏RNA在保存23个月后,5S条带较清晰,28S和18S条带均不清晰;胃部、肝脏及肾脏28S∶18S在0.34~0.72,肠道28S∶18S在0.43~2.15。高寒牧区牦牛胃肠道等组织按Trizol提取方法可提取高质量的RNA样品,但常规方法保存23个月的RNA样品均会产生不同程度的降解,降解程度亦存在动物基因型及组织部位的差异性。

The habitat and biological behavior of yaks are considerably different from those of indigenous cattle. It is not easy to obtain samples,in particular. The preservation method of the hard-won samples for long periods is important.Yaks and indigenous cattle served as tissue donors for developing an effective method for the storage and quality monitoring of RNA. Three yaks and cattle each,were slaughtered to obtain samples of gastrointestinal,liver,and kidney tissues,and RNA were extracted using a TRizol Kit; additionally,RNA concentration and the degree of RNA degradation were analyzed as per the Food and Drug Administration( FDA) regulations with Gel-pro Analyzer after the RNA samples were stored for 1 and 23 months at-80 ℃,respectively. The results indicated that after 23 months,RNA concentration increased in all the yak gastric tissue samples; however,it decreased in the rumina and omasa of the cattle. RNA was relatively stable in jejunal and ileal tissues of the yaks and cattle. There were significant differences in RNA concentration in the large intestine between the yaks and cattle( P < 0. 05). Gel-pro Analyzer results indicated that 5 S bands were clear; however,28 S and 18 S bands were vague in all the gastric,liver,and kidney tissues. The 28 S: 18 S ratio ranged from 0. 34 to 0. 72 for the gastric,liver,and kidney tissues: however,it ranged from 0. 43 to 2. 15 for intestinal tissues after storing for 23 months at-80 ℃. It was feasible to obtain RNA of high quality from the gastrointestinal samples of ruminants in the Qinghai-Tibet plateau. However,the RNA samples were degraded during storage by the conventional method. Additionally,the degradation degree was affected by several factors,such as genotype and tissue type.