A subset of 49 subjects had (nanograms per milligrams), with a ratio greater than 20 representing an index of thyrotoxicosis, as previously reported (19).All subjects underwent ultrasonographic evaluation of the thyroid, and the scans were reviewed by an experienced radiologist (T.
For each experiment 10 ng of c DNA samples were tested in duplicate.
The D2 promoter was previously cloned in p GL3 reporter vector (20), and the promoter-luciferase cassette was transferred into a pc DNA3.1 vector (pc DNA 3.1/D2pro-luc) carrying the G418 resistance gene for stable transfections selection.
Total RNA from normal thyroid (five subjects), toxic adenoma (three subjects), and MAS (five subjects) were extracted from tissue samples using the RNAzol method, and 500 ng of sample was converted to first-strand c DNA using a reverse transcriptase kit from Marligen (Ijamsville, MD) according to the manufacturer’s instructions.
Real-time PCR experiments were performed in triplicate and RNA levels were expressed as arbitrary units (AU) ± 1 gene encompassing, respectively, codons 201 and 228, were amplified from the thyroid c DNA samples using the following primers: 5′-TGAACGTGCCTGACTTTGAC-3′ (sense) and 5′-TCCACCTGGAACTTGGTCTC-3′ (antisense) and 5′-GCCCAGTACTTCCTGGACAA-3′ (sense) and 5′-ACCACGAAGATGATGGCAGT-3′ (antisense) using Platinum Taq polymerase (Invitrogen), and sequenced on both strands by Big Dye terminator version 3.1 using an ABI PRISM 3730XL analyzer (Applied Biosystems).
, the supernatant was collected and stored at −80 C.