Quantification of rat pre-pro-thyrotropin releasing hormone (TRH) mRNA by reverse transcription-polymerase chain reaction using external and internal standardisation

We have developed a quantitative reverse transcription polymerase chain reaction (RT-PCR) method, using both internal and external standardisation, to quantitate fg amounts of pre pro-thyrotropin releasing hormone (pre-pro-TRH) mRNA in the paraventricular nucleus (PVN) of individual laboratory rats. A constant amount of internal standard is coamplified with both the cDNA of each unknown specimen and a dilution series of an identical RT-PCR generated external standard, allowing quantitation of the samples by interpolation from the external standard curve. Pre-pro-TRH mRNA levels in the PVN were reduced by thyroxine (T-4) treatment to 48% of those in control animals, and were increased by thyroidectomy to 155% of the control levels. By combining external and internal standardisation, our method allows multiple samples and treatment groups to be assayed concurrently, thereby eliminating inter-assay variability, whilst retaining the advantages of internal standardisation. It will facilitate further studies of the control of TRH gene expression in pathophysiological conditions.