Dual fluorescence detection of protein and RNA in Drosophila tissues.
Dual fluorescence detection of protein and RNA in Drosophila tissues.
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DOI：
10.1038/nprot.2012.105
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发表时间：
2012-10
期刊：
影响因子：
14.8
通讯作者：
Jones DL
中科院分区：
文献类型：
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作者：
Toledano H;D'Alterio C;Loza-Coll M;Jones DL
通过原位杂交（ISH）检测RNA是一种成熟的技术，其允许研究组织中的特异性RNA表达模式;然而，并非所有组织都同样适合使用相同程序染色。在这里，我们描述了一种结合整体免疫荧光（IF）和荧光原位杂交（FISH）的方案，用于同时检测特定的RNA转录物和蛋白质，大大提高了复杂、完整苍蝇组织中RNA表达的空间分辨率。到目前为止，我们已经成功地使用该协议在成人睾丸，幼虫雄性性腺，成人肠和马氏管。在FISH的苛刻条件之前，在无RNA酶的溶液中进行IF，以保持解剖组织内的蛋白质抗原性。分别描述了用于mRNA和miRNA检测的单独方案，其分别基于稳健的地高辛（DIG）RNA和锁核酸（LNA）探针。联合IF-FISH检测miRNA和mRNA的时间分别为2d和4d。虽然针对果蝇进行了优化，但IF-FISH方案应适用于各种生物体、组织、抗体和探针，从而提供了一种可靠且简单的方法来比较RNA和蛋白质丰度和定位。
Detection of RNAs by in situ hybridization (ISH) is a well-established technique that permits the study of specific RNA expression patterns in tissues; however, not all tissues are equally amenable to staining using the same procedure. Here we describe a protocol that combines whole-mount immunofluorescence (IF) and fluorescence in situ hybridization (FISH) for the simultaneous detection of specific RNA transcripts and proteins, greatly enhancing the spatial resolution of RNA expression in complex, intact fly tissues. To date, we have successfully used this protocol in adult testis, larval male gonads, adult intestine and Malpighian tubules. IF is conducted in RNase-free solutions, prior to the harsh conditions of FISH, in order to preserve protein antigenicity within dissected tissues. Separate protocols are described for mRNA and miRNA detection, which are based on robust digoxigenin (DIG) RNA and locked nucleic acid (LNA) probes, respectively. The combined IF-FISH procedure can be completed in 2 d for miRNA detection and 4 d for mRNA detection. Although optimized for Drosophila, this IF-FISH protocol should be adaptable to a wide variety of organisms, tissues, antibodies and probes, thus providing a reliable and simple means to compare RNA and protein abundance and localization.