title: Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues.
abstract: OBJECTIVE
  The TSH receptor (TSHr) expressed on thyroid follicular cells is the autoantigen involved in the pathogenesis of Graves' hyperthyroidism. Whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of thyroid-associated ophthalmopathy (TAO) is unclear.
  
  
  METHODS
  The aim of the present study was to measure TSHr mRNA in retro-orbital tissues, retro-orbital adipose tissue, extraocular muscle, and skin from patients with TAO and in several tissues from patients not affected by thyroid diseases using RT-PCR and real-time PCR.
  
  
  METHODS
  Total RNA was isolated from tissue specimens, reverse transcribed, and amplified using specific primers for the extracellular portion and a part of a 1.3 kbp variant form of the TSHr gene. Determination of TSHr mRNA levels using real-time PCR was also performed by the TaqMan system; to normalize for differences in the amount of total RNA added to the reaction, amplification of beta-actin gene was performed as an endogenous control.
  
  
  RESULTS
  A single-round RT-PCR amplification using specific primers for the extracellular portion of the TSHr gene demonstrated an amplification product of 1.2 kbp in the thyroid, but not in all other tissues. A second-round RT-PCR amplification using the same primers and starting from the previous amplification product demonstrated a band of the size expected for the TSHr gene in all tissue specimens analyzed irrespective of their origin. Similar results were obtained using primers specific for a part of the variant form of 1.3 kbp of the TSHr gene. The amount of TSHr mRNA measured by real-time PCR with the TaqMan probe and expressed as TSHr/beta-actin ratio was similar in the tissues from TAO patients with respect to the tissues from subjects not affected by thyroid diseases.
  
  
  CONCLUSIONS
  We measured TSHr mRNA in tissues from patients with TAO using the very sensitive and quantitative method of real-time PCR. The level of transcription was similar to that measured in extraorbital tissues from patients who were not affected by thyroid diseases. These data suggest an illegitimate TSHr mRNA transcription in all the tissues examined apart from thyroid.
authors.affiliation: Dipartimento di Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, Universita di Pisa, Via Paradisa 2, 56124 Cisanello, Pisa, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Agretti
authors.forename: Luca
authors.initials: L
authors.lastname: Chiovato
authors.forename: Giuseppina
authors.initials: G
authors.lastname: De Marco
authors.forename: Claudio
authors.initials: C
authors.lastname: Marcocci
authors.forename: Barbara
authors.initials: B
authors.lastname: Mazzi
authors.forename: Stefano
authors.initials: S
authors.lastname: Sellari-Franceschini
authors.forename: Paolo
authors.initials: P
authors.lastname: Vitti
authors.forename: Aldo
authors.initials: A
authors.lastname: Pinchera
authors.forename: Massimo
authors.initials: M
authors.lastname: Tonacchera
mesh_terms: D000273:Adipose Tissue / Q000378:metabolism*; D003199:Computer Systems; D006801:Humans; D009801:Oculomotor Muscles / Q000378:metabolism*; D009915:Orbit / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D011989:Receptors, Thyrotropin