Glycoprotein Methods and Protocols - P25

In Situ Hybridization of Mucin mRNA 323

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In Situ Hybridization Techniques for Localizing Mucin mRNA

Ilene K. Gipson

1. Introduction

Progress in understanding how mucosal surfaces are protected is closely related to

the development of morphologic techniques to study the structure and secretory func￾tion of the mucosal epithelia. Morphologic methods have allowed characterization of

mucus-secreting cells of the epithelia of the eye, and the respiratory, gastrointestinal

(GI), and reproductive tracts. Characteristics of the mucus-secreting cells of these tis￾sues vary, and many questions remain regarding special characteristics of mucus

present over the differing mucosal surfaces. Recent progress in cloning and character￾ization of mucin genes has facilitated the use of in situ hybridization (ISH) to begin to

characterize the mucin gene repertoires and specific functions of mucins expressed by

the various epithelia, either those covering mucosal surfaces or glandular epithelia

contributing to the mucous layer on the surface of the tissue. ISH has been a particu￾larly valuable method in this regard, since antibodies to specific mucin proteins are

often difficult to use on tissues or secretions without heroic methods to deglycosylate

in order to make protein epitopes available.

Mucins, because of their heavy glycosylation and size, have presented major tech￾nical difficulties to biochemists and molecular biologists struggling to characterize

them (1–3). The use of molecular techniques to sequence the mucin gene has identi￾fied a characteristic common to all mucin genes, that of tandemly repeated sequences

in their amino acid/nucleotide sequence. (For review see refs. 4 and 5). This character

greatly facilitates application of ISH methods to localize specific mucin mRNAs in

tissues and cells. Probes to the tandemly repeated nucleotide sequences bind at mul￾tiple sites along cellular mRNA, providing an amplified signal and excellent visual￾ization of the presence of specific mucin mRNAs. For once, there is something about

mucin character that facilitates ease of application of a method! While this enhanced

signal is useful, it is an impediment to quantitative assays. One cannot rely on the use

of tandem repeat (TR) probes to quantitate mRNA levels, especially with those mucin

genes that exhibit polymorphisms.

From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ