直接原位逆转录聚合酶链反应

Direct in situ rt-PCR.

作者信息

Lossi Laura, Gambino Graziana, Salio Chiara, Merighi Adalberto

机构信息

Dipartimento di Morfofisiologia Veterinaria, Università degli Studi di Torino, Grugliasco, TO, Italy.

出版信息

Methods Mol Biol. 2011;789:111-26. doi: 10.1007/978-1-61779-310-3_6.

DOI:10.1007/978-1-61779-310-3_6

PMID:21922403

Abstract

In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.

摘要

原位聚合酶链反应（PCR）是一种组织学技术，它利用PCR的优势直接在组织切片中检测mRNA。它以某种方式将PCR与原位杂交结合在一起，而原位杂交传统上更多地用于在细胞器、完整细胞或组织切片中定位mRNA。本章描述了原位PCR在神经肽mRNA定位中的应用。我们在此提供了一种详细的方案，用于在固定和石蜡包埋或冷冻切片后，使用非放射性探针进行直接原位逆转录（RT）PCR（逆转录PCR）。在逆转录后，将地高辛标记的核苷酸（地高辛-11-dUTP）掺入PCR产物中，随后用与碱性磷酸酶偶联的抗地高辛抗体进行检测。该程序可进行修改以用于荧光探针，并可与酶/荧光免疫细胞化学标记结合使用。