Dual Color Detection of Splice Variants of the c-erbA α (Thyroid Hormone Receptor α) Gene

Abstract

Thyroid hormone (T₃) is important for many metabolic processes and signals its presence to the cell via nuclear thyroid hormone receptors (TR) [1]. There are two genes encoding for these receptors giving rise to four isoforms of the receptor (α1, α2, β1 and β 2). The TRα1 and TRα2 isoforms are the result of alternative splicing from the primary transcript of the c-erbA-α gene. The expression of these receptors changes as a result of various developmental and metabolic conditions. One of these is non-thyroidal illness, also called the sick-euthyroid syndrome (SES). It is characterized by an aberrant thyroid hormone metabolism in which the levels of the active hormone T₃ in the blood are lowered without an accompanying rise in TSH. The pathophysiology of this phenomenon is not clear, but cytokines are known to play a role. Furthermore, some liver genes (like malic enzyme) become refractory to exogenous T₃ as a result of the SES. This could be the consequence of an increased expression of the TRα2 isoform which can act as a dominant negative of the other three receptors. In the past, we have studied the expression of the isoform mRNAs in the liver and heart of fasting rats (a model of SES) and found that the level of the liver TRα2 mRNA indeed increases approximately threefold [2]. These results were obtained by using a semi-quantitative PCR technique based on competition between a standard and the target. With this technique the expression levels of the TRαl and -α2 mRNAs in one sample have to be measured in separate reactions, thereby introducing the possibility of errors. With the advent of the LightCyler we are now able to measure the TRα1 and -α2 mRNA levels (and/or the ratio) directly and specifically in one reaction, using hybridization probes and the dual color detection format. This chapter describes the approach we have developed to accomplish this.