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 (T4) 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.
Department of Clinical Chemistry, Queen Elizabeth Hospital, Woodville, South Australia, Australia.
L M
LM
Muller
J A
JA
Kennedy
D000818:Animals; D015139:Blotting, Southern; D008297:Male; D010286:Paraventricular Hypothalamic Nucleus / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D013973:Thyrotropin-Releasing Hormone