Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724, USA.
RNA. 2010 Mar;16(3):632-7. doi: 10.1261/rna.1775610. Epub 2010 Jan 19.
In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.
原位杂交广泛用于在胚胎、组织和细胞中可视化转录序列。对于胚胎中 mRNA 的全胚胎检测，通过与反义 RNA 探针杂交，然后对靶 mRNA 进行视觉或荧光检测。这种方法的一个限制是，必须获得靶 RNA 的 cDNA 模板，以便生成反义 RNA 探针。在这里，我们研究了使用短（12-24 个核苷酸）锁核酸（LNA）包含的 DNA 探针进行全胚胎原位杂交检测 mRNA。经过广泛的方案优化，我们表明 LNA 探针可用于定位鸡胚胎中几种丰度不同的 mRNA。LNA 探针还检测了以组织特异性方式进行加工的选择性剪接外显子。LNA 探针用于全胚胎原位检测 mRNA 将能够进行计算机设计和化学合成，并将扩大原位杂交在转录调控和选择性剪接研究中的一般用途。
