Synthesis and Purification of Digoxigenin-Labeled RNA Probes for In Situ Hybridization
This protocol was adapted from “Whole-Mount In Situ Hybridization,” Chapter 13, in Early Development of Xenopus laevis by Hazel L. Sive, Robert M. Grainger, and Richard M. Harland. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.INTRODUCTION
In situ hybridization is the most versatile method for determining when and where embryonic transcripts are expressed. Although a detailed analysis of gene expression often requires analysis of sectioned material, the whole-mount approach is invariably the first method used to localize gene expression. Whole-mount in situ hybridization has also become an invaluable tool for analyzing experimentally manipulated embryos and explants. The information gained using this technique is similar to that obtained from immunohistochemistry and includes not only the location of expression but a semiquantitative estimate of the relative levels of gene expression in different parts of the embryo. Because most genes are first analyzed as genomic or cDNA clones, it is straightforward to derive specific probes from these sequences. In this protocol, standard RNA synthesis using a bacteriophage polymerase is carried out incorporating a digoxigenin-substituted ribonucleotide, dig-UTP. In vitro transcription in the direction opposite to in vivo transcription of the mRNA makes an antisense RNA that is complementary to the mRNA. The ratio of dig-UTP to UTP has been optimized to give efficient synthesis and efficient detection of the probe. The three commercially available bacteriophage polymerases (SP6, T7, and T3) all incorporate the substituted nucleotide with equivalent efficiencies, and thus there is a wide choice of plasmid vectors to prepare the template.
