In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha).
In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha).
复制标题
使用延伸因子 1 α (EF-1 α) 对异质组织样品中 mRNA 可及性进行原位评估。
DOI：
10.1007/pl00007903
复制
发表时间：
1997
影响因子：
2.3
通讯作者：
Levine,RA
中科院分区：
文献类型：
--
作者：
Gruber,AD;Levine,RA
延伸因子-1α (EF-1α) 是一种进化上高度保守的所有活细胞中蛋白质合成的通用辅助因子。在这项研究中，评估了其作为原位杂交检测 mRNA 序列特异性检测的阳性对照的用途。对不同汇合阶段、细胞形状和细胞周期状态的各种非肿瘤性和肿瘤性培养细胞的 Northern 印迹分析表明，EF-1α 的表达比 β-肌动蛋白更低且更均匀。使用地高辛标记的核糖探针在常规福尔马林固定、石蜡包埋的组织切片中检测 EF-1α mRNA 的原位杂交测定表明，EF-1α 是所有类型细胞中合适的阳性对照。然而，蛋白酶预处理的变化表明同一组织样品中不同细胞类型的消化条件不同，有时甚至是相互排斥的。我们的结果表明，EF-1α mRNA 的检测是原位杂交测定的适当内标，并且有助于控制伪影，例如由不适当的蛋白酶预处理引起的假阴性。观察到的同一组织切片内不同细胞类型的最佳蛋白酶预处理的可变性增强了原位杂交测定中阳性对照的重要性。
Elongation factor-1α (EF-1α) is an evolutionarily highly conserved universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assays for specific detection of mRNA sequences was evaluated. Northern blot analysis of various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1α had a lower and more homogeneous expression than did β-actin. In situ hybridization assays using digoxigenin-labeled riboprobes for the detection of EF-1α mRNA in routinely formalin-fixed, paraffin-embedded tissue sections showed that EF-1α is a suitable positive control in all types of cells. However, variation of protease pretreatments demonstrated distinct and sometimes mutually exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1α mRNA is an appropriate internal standard for in situ hybridization assays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variability of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive control in in situ hybridization assays.