Degradation of intestinal mRNA: A matter of treatment
Degradation of intestinal mRNA: A matter of treatment
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DOI：
10.3748/wjg.v21.i12.3499
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发表时间：
2015-03-28
影响因子：
4.3
通讯作者：
Schaefer, Karl-Herbert
中科院分区：
文献类型：
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作者：
Heumueller-Klug, Sabine;Sticht, Carsten;Schaefer, Karl-Herbert
目的：研究不同部位、不同种类和不同处理方式对胃肠道组织标本RNA降解的影响。方法：不同种类(人和大鼠)、不同温度(冰块或常温保存)、粘膜下层剥离时间(保存前或保存后)、不同冲洗方法(用介质、PBS、RNAlAla冲洗或不冲洗)和肠道不同部位(小肠近端和远端、盲肠、结肠和直肠)的不同时间保存。提取肠道不同部分(大鼠：近端和远端小肠、盲肠、结肠和直肠，人：结肠和直肠)和单个肠层(肌肉和粘膜下/粘膜)的总RNA。采用微量毛细管电泳法对提取的RNA进行质量评价。用18个S和28个S RNA峰的相对高度和相对面积计算的RNA完整性数来表示RNA质量。结果：从大鼠远端小肠组织中获得的RNA比从粘膜下/粘膜组织中获得的RNA稳定时间更长。在RT时，肌肉RNA在一天后降解，在冰上至少稳定三天。在储存和使用RNAlater之前清洁和分离肠道层，可以在更长的时间内保持肌肉RNA在RT下的稳定性。肠道的不同部分表现出不同的降解期。从粘膜下层/粘膜层获得的RNA的扩增率总是比从肌肉组织获得的RNA差得多。一般来说，从大鼠组织中获得的RNA无论是从平滑肌层还是从粘膜下/粘膜层获得的RNA都比来自人类肠道组织的RNA稳定得多，从平滑肌肉组织中获得的RNA比从粘膜下/粘膜组织中获得的RNA显示出更高的稳定性。在RT时，肌肉RNA在一天后降解，而在冰上的稳定性至少持续三天。在储存和使用RNAlater之前清洁和分离肠道层，可以在更长的时间内保持肌肉RNA在RT下的稳定性。肠道的不同部分表现出不同的降解期。来自近端小肠肌肉和粘膜下/粘膜组织的RNA降解速度远快于远端小肠、盲肠或直肠结肠的RNA。从粘膜下层/粘膜层获得的RNA的扩增率始终比从肌肉组织获得的RNA的扩增率低得多[β-微管蛋白III的肌肉量化周期(C-p)：22.07+/-0.25，粘膜下/粘膜层的β-微管蛋白III的C-p：27.42+/-0.19]。结论：肠道mRNA的降解取决于组织的制备和保存条件。肠道组织的冷却、冲洗和分离减少了mRNA的降解。
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 varying conditions: 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 qPCR 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 colon with rectum. RNA obtained from the submucosal/mucosal layer always showed a much more reduced amplification rate than RNA from muscle tissue [beta-Tubulin III for muscle quantification cycle (C-p): 22.07 +/- 0.25, for beta-Tubulin III submucosal/mucosal C-p: 27.42 +/- 0.19].CONCLUSION: Degradation of intestinal mRNA depends on preparation and storage conditions of the tissue. Cooling, rinsing and separating of intestinal tissue reduce the degradation of mRNA.