Detection of Circulating Thyroid Cancer Cells by Reverse Transcription-PCR for Thyroid-stimulating Hormone Receptor and Thyroglobulin: The Importance of Primer Selection

Gupta, Manjula K.; Taguba, Leslie; Arciaga, Rosemary; Siperstein, Allan; Faiman, Charles; Mehta, Adi; Reddy, S. Sethu K.

doi:10.1093/clinchem/48.10.1862

Cited by 29 publications

(7 citation statements)

References 8 publications

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“…[13] in their research, used several primer pairs of different exons. They found that primer pairs for TSHR targeting 6 to 9 exons and 1 to 5 exons for Tg , were with specificity for thyroid tissue and no reactivity in normal peripheral blood mononuclear cells excluding possible illegitimate transcription [13,14]. In our study, we used the same primer pairs as in the study of Gupta et al .…”

Section: Discussionmentioning

confidence: 99%

“…In our study, we used the same primer pairs as in the study of Gupta et al . [13]. On the other hand, Savagner et al .…”

Section: Discussionmentioning

confidence: 99%

“…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 Тg- 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).…”

Section: Methodsmentioning

confidence: 99%

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

Makazlieva

Eftimov²,

Vaskova

et al. 2018

Balkan Journal of Medical Genetics

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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.

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Section: Discussionmentioning

confidence: 99%

“…In our study, we used the same primer pairs as in the study of Gupta et al . [13]. On the other hand, Savagner et al .…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Makazlieva

Eftimov²,

Vaskova

et al. 2018

Balkan Journal of Medical Genetics

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“…Total RNA was isolated from 3 mL EDTA blood using the Geneaid kit (Geneaid, Taiwan) according to the manufacturer’s protocol. Quantitative PCR was performed using 4 μL of first-strand cDNA in a 20- l reaction volume containing 0.2 μM of each TSHR primer (Forward primer 5′-GTTCCCTGACCTGACCAAAG-3′and Reverse primer 5′-AAGGGCAGTGACACTGGTTTGAGA-3′[ 14 ]) and 10 μL of SensiFast SYBR no ROX (Bioline, MA); cycling conditions for PCR included denaturing for 2 min at 95 °C; followed by 40 cycles of 110 s at 95 °C; 15 s at 60 °C; 20 s at 72 °C; and 20 s at 72 °C; using LightCycler@480 Real-Time PCR System (Roche Diagnnostics GmbH, Germany). Each sample was assayed in duplicate.…”

Section: Methodsmentioning

confidence: 99%

Circulating thyrotropin receptor messenger ribonucleic acid is not an effective marker in the follow-up of differentiated thyroid carcinoma

et al. 2015

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BackgroundCirculating thyrotropin receptor messenger ribonucleic acid (TSHR mRNA) assay has been validated in the follow-up of differentiated thyroid carcinoma (DTC) because of its high sensitivity during thyroid hormone therapy and no interference with endogenous anti-thyroglobulin antibodies (TgAb) compared to serum thyroglobulin (Tg). We investigated the efficacy of TSHR mRNA assay in 160 DTC patients using quantitative PCR (qPCR).FindingsOnly TSHR mRNA level of structural persistent disease with TgAb-positive (3.47 (2.97–9.53) pg equivalents/μg total RNA; p = 0.013) and its subgroup of distant metastasis patients with TgAb-positive (5.55 (3.28–12.52) pg equivalents/μg total RNA; p = 0.009) were significantly different from patients with no evidence of disease (2.32 (1.44–3.94) pg equivalents/μg total RNA). Applying cutoff at 2.00 pg equivalents/μg total RNA enabled us to predict structural persistent disease patients with a sensitivity of 62.3 % and a specificity of 42.9 %. Although, the sensitivity of TSHR mRNA assay in TgAb-postive patients (88.2 %) was superior than serum Tg (47.1 %) (p = 0.00002), the accuracy of the test is only 54.5 %.ConclusionsThis study demonstrated that TSHR mRNA assay has good sensitivity in TgAb-positive patients but it is neither specific enough as a first-line of testing nor a surrogate marker in the follow-up of our DTC patients.

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“…Many studies on circulating tumor cells have been performed, especially in patients with breast cancer, malignant melanoma and prostate adenocarcinoma; however, in thyroid carcinoma, these steps are still incipient [ 31 , 32 ]. This review reflects the concerns and interests in understanding the processes of metastasis in thyroid carcinoma, pathology with an increasing incidence worldwide [ 33 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

Circulating tumour cells in thyroid carcinoma - the prognostic role of this biomarker. Review of the literature.

Bădulescu

Bărbuş

Piciu

2017

Medicine and Pharmacy Reports

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Thyroid cancer is a disease with a good prognosis and high survival rates, but having a marked growth of incidence all over the world in the last years. This fact requires special attention of researchers for understanding the behavior of this disease and to establish a correct therapy. Analysis of circulating tumor cells in patients with different malignancies is nowadays a new and exciting research tool, which can improve the diagnosis and prevent the metastatic disease. In the case of thyroid carcinoma there are few studies which explore these biomarkers and investigate the prognostic significance of circulating tumor cells. With this review we seek to emphasize the role of these cells to better understand the mechanisms of invasion or metastasis and to establish a new research base to treat aggressive forms of this type of cancer. Most of the included studies demonstrate the efficacy of these markers for diagnosis and follow up.

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Detection of Circulating Thyroid Cancer Cells by Reverse Transcription-PCR for Thyroid-stimulating Hormone Receptor and Thyroglobulin: The Importance of Primer Selection

Cited by 29 publications

References 8 publications

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

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

Circulating thyrotropin receptor messenger ribonucleic acid is not an effective marker in the follow-up of differentiated thyroid carcinoma

Circulating tumour cells in thyroid carcinoma - the prognostic role of this biomarker. Review of the literature.

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