Localization of RNA Expression

Authored by: Scott A. Harding , Chung-Jui Tsai , Leland J. Cseke

Handbook of Molecular and Cellular Methods in Biology and Medicine

Print publication date:  December  2011
Online publication date:  April  2016

Print ISBN: 9781420069389
eBook ISBN: 9781439881958
Adobe ISBN:

10.1201/b11351-17

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Abstract

Hybridization of paraffin- or plastic-embedded tissue sections with labeled probes to visualize the cellular distribution of mRNA transcripts is known as in situ hybridization (ISH). The following ISH protocol is based on that of Jackson, 1 but it includes a number of modifications to improve results from woody plant tissues. Special features of this protocol include the use of full-length RNA probes at high hybridization temperatures, followed, when colorimetric detection is used, by color development in buffer containing polyvinyl alcohol (PVA) to reduce chromagen diffusion. 2 These modifications reduce background from nonspecific hybridization and reduce color development time while improving signal localization. The protocol works well with soft tissues and eliminates background due to nonspecific probe sticking in secondary vascular tissues of woody plant specimens. The method is applicable to animal tissues as well. RNase-free technique (see Chapter 8) should be employed throughout. Procedural modifications for performing ISH with [35S]-labeled probes 1 , 3 , 4 are included where appropriate. However, most steps of the ISH protocol are applicable to both detection systems.

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