Nonradioactive In Situ Hybridization for Cells and Tissues

In situ hybridization (ISH) is a widely used technique that has great power in many applications, including diagnosis of viral infections (1 ), chromosome analysis (2 ), and mRNA analysis (3 ). Traditionally, researchers have used radioactive labels to prepare probes for ISH (4 ). Such probes can be labeled DNA, RNA, or oligonucleotides. RNA and oligonucleotide probes are most often used because they have several features of advantage for ISH (see Table 1 ); in particular, they are single stranded, offering high sensitivity (RNA) and high-specificity (oligonucleotide). Recently, nonradioactive probes have become more popular, and the same features hold true for these. In this chapter, the ISH process is illustrated by use of a system for the detection of mRNA on tissue sections with RNA probes, but it is equally applicable to work on cell lines and for DNA detection in such systems. In addition, the detection procedures described with the in situ process are applicable to RNA, DNA, and oligonucleotide probes. The analysis of chromosomes and nuclei is a distinct procedure that utilizes nonradioactive DNA probes and fluorescent detection (5 ).

	Features
Probe type	Positive	Negative
RNA probes	RNA hybrids have a higher stability	Subcloning required
	Single-stranded	RNase degradation possible
	No vector sequences present	Critical hybridzation conditions
	Controls easy to produce
	Low background
	High sensitivity
Oligonucleotides	Single-stranded	Low level of labeling
	Highly specific	Sequence optimization required
	Easy access to target	Cocktails of probes may be needed
	Stable
	Easy to produce m large amounts
DNA probes	No subcloning required	Probe denaturation required
	Easy and reliable labeling methods	Probe reanneals in hybridization
	Less critical hybridization conditions	Difficult to remove vector sequences