In situ hybridization detection of calcitonin mRNA in routinely fixed, paraffin-embedded tissue sections: a comparison of different types of probes combined with tyramide signal amplification

Abstract

In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids in tissues and cells. However, its use is limited by the relative lack of sensitivity in detecting low copy numbers of nucleic acids. Several strategies have been developed to improve the threshold levels of in situ detection of nucleic acid by amplification of either target nucleic acid sequences before ISH (such as in situ polymerase chain reaction) or after the hybridization procedure (such as tyramide signal amplification [TSA]). The authors compared the use of different types of probes to detect calcitonin mRNA in 10 cases of formalin-fixed, paraffin-embedded medullary thyroid carcinoma with and without TSA. In addition, dot blot hybridization was used to compare the signal amplification by TSA with oligonucleotide. cDNA, and cRNA probes. These results show that cRNA probes are the most sensitive types of probes for detecting mRNA molecules in formalin-fixed, paraffin-embedded tissue sections and that tyramide amplification can increase the sensitivity for detection of calcitonin mRNA in formalin-fixed, paraffin-embedded tissue sections at least 2- to 4-fold with cRNA probes.