Balkan Journal of Medical Genetics

DETERMINING SPECIFIC THYROID TRANSCRIPTS IN PERIPHERAL BLOOD: A SINGLE CENTER STUDY EXPERIENCE
Makazlieva T, Eftimov A, Vaskova O, Tripunoski T, Miladinova D, Risteski S, Jovanovic H, Jakovski Z Tanja Makazlieva and Aleksandar Eftimov contributed equally to this study and are considered first coauthors.
*Corresponding Author: Tanja Makazlieva, Ph.D., Institute of Pathophysiology and Nuclear Medicine, Medical Faculty, Mother Teresa Street, No. 17, 1000, Skopje, Republic of Macedonia. Mobile: +389-75-313-665. E-mail: tmakazlieva@medf.ukim.edu.mk or tmakazlieva@gmail.com
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MATERIALS AND METHODS

Subjects. The study included blood sampling from a total of 57 subjects, including 40 patients with DTC and 17 healthy volunteers as a control group. The inclusion criteria for the control group were normal isoechoic appearance of the thyroid gland and absence of nodules at ultrasound examination (US) of the neck (linear transducer 7.5-10 MHz), normal blood levels for thyroid stimulating hormone (TSH) and free thyroxine (FT4), and antithyroid antibodies that are below the level usually considered to be clinically significant. In all patients from the DTC group, the surgery and ablation treatment with radioactive 131I were conducted at least 6 months before starting the research. In all DTC patients blood samples for RNA extraction were drawn prior to US neck examination. Similar to the concept of risk stratification during follow up introduced by Tuttle et al. [12], DTC patients were divided in three subgroups. The three groups of patients were selected according to blood levels of sTg (CRM 457, Immulite 2000; Siemens, Munich, Bavaria, Germany) and aTg, as well as on the US neck examination and findings from WBS after ablation with 131I or diagnostic WBS. The first group consisted of patients with incomplete structural response to treatment and biochemical relapse of the disease, imaging confirmed the metastatic spread or loco-regional relapse and elevated sTg levels, >0.2 ng/mL (TCs 22 patients). The second group were patients with incomplete biochemical response and indeterminate response, with only elevated sTg (>0.2 ng/mL), or elevated aTg levels, without confirmed and known morphological signs for relapse (TCb six patients). The third group of patients were with complete structural and biochemical response to therapy, excellent responders (TCr 12 patients). Corresponding to the histopathological type from 40 patients, four cases were FTC, 25 typical variants of PTC, one PTC in thyroglossal duct cyst, one papillary microcarcinoma, seven follicular variants of PTC and two Hurtle cell carcinomas. The study was approved by the Ethics Committee at the Medical Faculty of the University “Ss Cyril and Methodius,” Skopje, Republic of Macedonia, and written consent was obtained from all subjects enrolled in the research. RNA Extraction Procedure and Real Time-Polymerase Chain Reaction. Whole blood samples were drawn from HC and TC patients in standard 3 mL EDTA blood vacutainers, total RNA was extracted using commercially available RNA isolation kit GenElute™ Total RNA purification kit (Sigma Aldrich Co. LLC, St. Louis, MO, USA) and isolated total RNA was used for two step RTPCR with ReadyScript™ cDNA Synthesis Mix kit (Sigma Aldrich), according to the manufacturer’s protocols. Real Time Polymerase Chain Reaction. The PCR step was performed using the following primer pairs: TSHR-F 5’-GCT TTT CAG GGA CTA TGC AAT GAA- 3’ and TSHR-R 5’-AAG GGC AGT GAC ACT GGT TTG AGA-3’, targeted to amplify a segment spanning exons 6 to 9 (nucleotides 555-767 or 212 bp) and Òg- F 5’-AGG GAA ACG GCC TTT CTG AA-3’ and Tg-R 5’-GTG GAG AAG ACG ACG ATT TC-3’, targeted to exons 1 to 5 (nucleotides 112-519 or 407 bp) [9]. The ubiquitously expressed GAPDH gene was used to confirm RNA extraction and RT-PCR using primers GAPDH-F 5’-TTC GTC ATG GGT GTG AAC C-3’ and GAPDH-R 5’-GAT GAT GTT CTG GAG AGC CC-3’, as previously reported [13,14]. For RT quantitative PCR (qPCR), we used Hot FirePol Eva Green qPCR Mix Plus (Rox) PCR master mix (Solis BioDyne, Tartu, Estonia). The reaction mixture was incubated at 95 °C for 15 min., followed by 38 cycles of denaturation at 95 °C for 15 seconds, annealing at 62 °C for 20 seconds and elongation at 72 °C for 20 seconds, for TSHR and Tg and incubated at 95 °C for 15 min., followed by 38 cycles of denaturation at 95 °C for 15 seconds, annealing at 57 °C for 20 seconds and elongation at 72 °C for 20 seconds, for GAPDH. All samples were analyzed in triplicate. Relative Quantification Method. Relative quantification was applied by calculating fold change in gene expression of the TSHR and Tg target genes, normalized to the endogenous reference gene GAPDH. Cycle threshold (Ct), ΔCt, ΔΔCt and normalized relative expression ratio values, were calculated according to the 2–ΔΔCt method, by Livak and Schmittgen [15]. According to this method, ΔCtTSHR = (average CtTSHR-average CtGAPDH), also for ΔCtTg = (average CtTg-average CtGAPDH) for every TC patient and HC was calculated, and then ΔΔCt = [ΔCt(TC)- ΔCt (HC)] was calculated from the average values of four groups and later normalized expression ratio 2–ΔΔCt was applied for fold change evaluation [15,16]. Statistically significant differences among all groups were evaluated with the Mann-Whitney U and Kruskal-Wallis H tests.

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