Quantitative reverse transcription-PCR measurement of thyroglobulin mRNA in peripheral blood of healthy subjects

Background: Thyroglobulin mRNA can be detected qualitatively in the periphe ral blood of patients with metastatic thyroid cancer, thyroid cancer patien ts with residual thyroid bed uptake, and individuals with no known thyroid disease with intact thyroid glands by use of a lengthy, highly sensitive ex traction technique. To improve and broaden the clinical usefulness of this assay, we developed a quantitative reverse transcription (RT)-PCR assay for thyroglobulin mRNA, using RNA recovered from whole blood with a simplified extraction technique. Methods: Whole blood was drawn from 32 healthy subjects in standard EDTA bl ood collection tubes. Total RNA was extracted from whole blood, using the P URE-script RNA isolation Kit. RT-PCR using intron-spanning primers was used to quantitatively amplify thyroglobulin mRNA, using the ABI PRISM 7700 Seq uence Detection System with a fluorescent-labeled, thyroglobulin-specific o ligonucleotide probe. Thyroid RNA calibration curves were created using tot al RNA recovered from a single nondiseased thyroid gland. Results: Qualitative RT-PCR demonstrated the presence of thyroglobulin mRNA in the whole blood sample of each healthy subject. The mean concentration of thyroglobulin mRNA detected in these subjects was 433 +/- 69 ng of total thyroid RNA per liter of whole blood (range, 26-1502 ng/L). Overall assay imprecision (CV) was 24% for five samples analyzed 10 times each in separat e analytical runs on different days. Conclusions: Thyroglobulin mRNA can be accurately detected and quantified i n peripheral blood from healthy subjects. This new quantitative technique m ay improve the clinical utility of circulating thyroglobulin mRNA detection in patients with thyroid disease. (C) 1999 American Association for Clinic al Chemistry.