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| 1 | Degradation of intestinal mRNA:A matter of treatment显示文摘AIM: To characterize the influence of location, species and treatment upon RNA degradation in tissue samples from the gastrointestinal tract. METHODS: The intestinal samples were stored in different medium for different times under varyingconditions: different species(human and rat), varying temperature(storage on crushed ice or room temperature), time point of dissection of the submucous-mucous layer from the smooth muscle(before or after storage), different rinsing methods(rinsing with Medium, PBS, RNALater or without rinsing at all) and different regions of the gut(proximal and distal small intestine, caecum, colon and rectum). The total RNA from different parts of the gut(rat: proximal and distal small intestine, caecum, colon and rectum, human: colon and rectum) and individual gut layers(muscle and submucosal/mucosal) was extracted. The quality of the RNA was assessed by micro capillary electrophoresis. The RNA quality was expressed by the RNA integrity number which is calculated from the relative height and area of the 18 S and 28 S RNA peaks. From rat distal small intestine q PCR was performed for neuronal and glial markers.RESULTS: RNA obtained from smooth muscle tissue is much longer stable than those from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, on ice it is stable at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. RNA obtained from the submucosal/mucosal layer always showed a much worse amplification rate than RNA from muscle tissue. In general RNA harvested from rat tissue, either smooth muscle layer or submucosal/mucosal layer is much longer stable than RNA from human gut tissue, and RNA obtained from smooth muscle tissue shows an increased stability compared to RNA from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, while the stability on ice lasts at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. The RNA from muscle and submucosal/mucosal tissue of the proximal small intestine degrades much faster than the RNA of distal small intestine, caecum or colonwith rectum. RNA obtained from the submucosal/mucosal layer always showed a much more reduced amplification rate than RNA from muscle tissue [β-Tubulin Ⅲ for muscle quantification cycle(Cp): 22.07 ± 0.25, for β-Tubulin Ⅲ submucosal/mucosal Cp: 27.42 ± 0.19].CONCLUSION: Degradation of intestinal m RNA depends on preparation and storage conditions of the tissue. Cooling, rinsing and separating of intestinal tissue reduce the degradation of m RNA. | Sabine Heumüller-Klug Carsten Sticht Karin Kaiser Elvira Wink Cornelia Hagl Lucas Wessel Karl-Herbert Schafer | 2015 | World Journal of Gastroenterology2015，21，12： | 2 |
| 2 | 大肠杆菌重组表达截短的人ACE2基因显示文摘利用E.coli原核表达系统克隆、重组表达截短的人血管紧张素转化酶2(ACE 2)。通过细胞分子生物技术获取截短的人ACE2的基因,并将其克隆到p ET28a载体上,且在大肠杆菌BL21(DE3)中重组表达该截短的人ACE 2蛋白。获取的截短的人ACE2基因,通过Nco I/Xho I双酶切后,将其克隆到经过同样Nco I/Xho I双酶切的p ET28a表达载体上,得到p ET28a-h ACE2重组质粒。然后将p ET28a-h ACE2重组质粒转化到大肠杆菌BL21(DE3)中,在37℃1 mmol/L IPTG诱导下重组表达了截短的人ACE2,通过免疫学方法初步鉴定正确表达了该ACE2。在原核表达系统中,重组表达截短的人ACE2;为研究ACE2的结构与功能的关系奠定了一定研究基础。 | 牛佳 师帆 | 2014 | 药物生物技术2014，21，5： | 0 |
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