Regulation of sodium-iodide-symporter gene expression in human thyrocytes measured by real-time polymerase chain reaction.
The sodium-iodide-symporter (NIS) plays a key role in iodination, the first step in the biosynthesis of the thyroid hormones, and is thought to be critically involved in several thyroid disorders associated with altered iodine up-take. To elucidate the pathogenic role of NIS in these diseases a sensitive technique is needed to measure human NIS gene expression. We established a real time RT-PCR for accurate quantification of hNIS mRNA levels based on fluorescence-labelled hybridisation probes in the LightCycler system. Human NIS expression was investigated in primary cultures of human thyrocytes. After optimisation of PCR conditions less than 10 molecules hNIS were detected with high sensitivity, specificity and reproducibility. Under basal conditions NIS expression varied from 83 to 593 copies per 10 6 GAPDH molecules. Stimulation of thyrocytes with TSH (0.1-10 U/ml) or forskolin (0.1-15 microM) results in a dose- and time-dependent up-regulation of NIS expression reaching a maximum at 10 mU/ml TSH (2211 +/- 761 copies) or 10 microM forskolin (1663 +/- 302 copies) after 24 h. In conclusion, we here established a real-time RT-PCR combining the advantages of rapid thermocycling and online detection of NIS mRNA amplification. The sensitive quantification of human NIS mRNA expression offered by this novel technique may improve analysis of hNIS regulation and measurement of NIS mRNA expression in small biopsies.
Wagner
S
S
German Diabetes Research Institute at the University of Duesseldorf, Germany.
Aust
G
G
Schott
M
M
Scherbaum
W A
WA
Feldkamp
J
J
Seissler
J
J
D001706:Biopsy; D015152:Blotting, Northern; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006042:Goiter / Q000378:metabolism; D006801:Humans; D012313:RNA; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters / Q000235:genetics*; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology; D000099209:Sodium-Iodide Symporters
D018076:DNA, Complementary; D012313:RNA; D012333:RNA, Messenger; D027981:Symporters; D013972:Thyrotropin; D000099209:Sodium-Iodide Symporters; C034477:RNA primers