Sensitive nonradioactive detection of mRNA in tissue sections: Novel application of the whole-mount in situ hybridization protocol
Sensitive nonradioactive detection of mRNA in tissue sections: Novel application of the whole-mount in situ hybridization protocol
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DOI：
10.1177/002215540104900101
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发表时间：
2001-01-01
影响因子：
3.2
通讯作者：
Christoffels, VM
中科院分区：
文献类型：
--
作者：
Moorman, AFM;Houweling, AC;Christoffels, VM
与染色体扩散中的单拷贝DNA序列或整装样品中的mRNA序列的灵敏非放射性检测相比，组织切片中的非放射性mRNA检测的相对不灵敏性仍然是一个令人困惑的问题。由于灵敏的原位mRNA检测结合高空间分辨率的生物学意义，我们开发了一种非放射性原位杂交（ISH）协议检测mRNA序列的部分。该程序基本上是基于整个安装ISH程序，至少是同样敏感。将杂交温度提高到70 ℃，同时通过调整盐浓度来保持杂交的严格性，显著提高了灵敏度，使该方法比常规放射性方法更灵敏。使用传统放射性ISH方案没有改善的较厚切片进一步增强了信号。较高的杂交温度显然允许探针更好地穿透组织。这种高度可靠的方案的应用允许在发育中的心脏中鉴定和定位细胞，这些细胞表达易洛魁同源盒基因家族不同成员的低丰度mRNA，这些基因家族被认为参与心脏模式。放射性ISH程序几乎不允许检测这些序列，这强调了这种新方法的价值。
The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.