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. Design: 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 β-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/β-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.
| Original language | English |
|---|---|
| Pages (from-to) | 733-739 |
| Number of pages | 7 |
| Journal | European Journal of Endocrinology |
| Volume | 147 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - Dec 1 2002 |
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ASJC Scopus subject areas
- Endocrinology
Cite this
Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues. / Agretti, Patrizia; Chiovato, Luca; De Marco, Giuseppina; Marcocci, Claudio; Mazzi, Barbara; Sellari-Franceschini, Stefano; Vitti, Paolo; Pinchera, Aldo; Tonacchera, Massimo.
In: European Journal of Endocrinology, Vol. 147, No. 6, 01.12.2002, p. 733-739.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Real-time PCR provides evidence for thyrotropin receptor mRNA expression in orbital as well as in extraorbital tissues
AU - Agretti, Patrizia
AU - Chiovato, Luca
AU - De Marco, Giuseppina
AU - Marcocci, Claudio
AU - Mazzi, Barbara
AU - Sellari-Franceschini, Stefano
AU - Vitti, Paolo
AU - Pinchera, Aldo
AU - Tonacchera, Massimo
PY - 2002/12/1
Y1 - 2002/12/1
N2 - 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. Design: 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 β-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/β-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.
AB - 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. Design: 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 β-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/β-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.
UR - http://www.scopus.com/inward/record.url?scp=0036924749&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036924749&partnerID=8YFLogxK
U2 - 10.1530/eje.0.1470733
DO - 10.1530/eje.0.1470733
M3 - Article
C2 - 12457447
AN - SCOPUS:0036924749
VL - 147
SP - 733
EP - 739
JO - European Journal of Endocrinology
JF - European Journal of Endocrinology
SN - 0804-4643
IS - 6
ER -