Determining specific thyroid transcripts in peripheral blood: A single center study experience

Abstract

Thyroid carcinoma (TC) comprises a spectrum of different tumors with a wide range of biological behavior and prognosis. The techniques based on the latest trends in molecular biology may have application in diagnosis of metastatic TC. The aim of this study was to apply and analyze mRNA expression in peripheral blood of thyrotropin receptor [thyroid stimulating hormone receptor (TSHR-mRNA)] gene and thyroglobulin (Tg-mRNA) gene using 2–ΔΔCt method in differentiated TC patients and healthy individuals. Fifty-seven subjects were included in the study, consisting of 40 patients with TC and 17 healthy volunteers as a control group. Total RNA was isolated from peripheral blood and used for two-step reverse transcriptase-polymerase chain reaction (PCR). Real-time PCR was performed with appropriate primers. Relative quantification using the 2–ΔΔCt method was applied. Thyroid carcinoma patients with metastatic disease or loco-regional relapse expressed TSHR-mRNA by a 8.57-fold higher level than healthy controls. Thyroid carcinoma patients with biochemical relapse expressed TSHR-mRNA by a 14.17-fold higher level than healthy controls, while expression of Tg-mRNA was 6.6-fold higher in TC patients with metastatic disease and loco-regional relapse than healthy controls and 8.34-fold higher level compared with TC patients with excellent response to treatment. Our preliminary study showed that the TSHR gene expression might have more useful application as a biomarker compared to detection of Tg gene expression.

Thyroid Stimulating Hormone Receptor Transcripts in Correlation with Clinical Parameters in Thyroid Carcinoma Patients

“…Descriptive analysis of all 40 patients is presented in Tables 1-3. We detected that TCs patients expressed mRNA-TSHR by a 5.37-fold higher level than TCr patients, TCb patients expressed mRNA-TSHR by an 8.88-fold higher level than TCr patients, as was previously reported in our previous study [19]. Further intergroup comparison between average ΔCt = (TSHR-GAPDH) values was performed and a significant difference was detected between TCs and TCr group (Mann-Whitney U-test: Z = −2.231, p 0.026) and TCb and TCr group (Mann-Whitney U-test: Z = −2.341, p = 0.019) (Figure 1).…”

“…Wagner et al [21] using RT-PCR detected a higher level of mRNA-TSHR in patients with benign thyroid nodules. We applied the semi-quantitative analysis using 2 -ΔΔCt method by Livak [14] in the previous research including healthy individuals and DTC patients and detected significantly higher expression in TCs and TCb group of patients compared to healthy control group [19]. Our further analysis was aimed at correlation of expression of mRNA-TSHR with sTg values, initial staging, US/ WBS findings and total received RAI therapy.…”

“…Cycle threshold (Ct), ΔCt, ΔΔCt, and normalized relative expression ratio values were calculated according to 2 -ΔΔCt method, by Livak and Schmittgen [14]. According to this method, ΔCtTSHR = (average CtTSHR-average CtGAPDH) and then ΔΔCt value was calculated using ΔCt value from healthy volunteers (HC), from data published previously by our group [19], or ΔΔCt = [ΔCt(TC)-ΔCt (HC)] and later normalized expression ratio 2 -ΔΔCt was applied for fold change evaluation [14], [15], [19].…”