Development of a Sensitive and Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Blood Thyroglobulin Messenger Ribonucleic Acid in the Follow-Up of Patients with Differentiated Thyroid Carcinoma

Context: Serum thyroglobulin is a sensitive tumor marker in the follow-up of patients with differentiated thyroid carcinoma (DTC), but the presence of endogenous anti-thyroglobulin antibodies (TgAb) can interfere on its measurement. To prevent interference by TgAb, several investigators have tried to quantify blood mRNA Tg by real-time RT-PCR, but the results have been variable, not reporting a correlation between mRNA Tg and the presence of metastases.Objective: the aim of the study was to evaluate the development of a sensitive and specific quantitative RT-PCR assay for blood mRNA Tg in the follow-up of patients with DTC.Design and Patients: An assay employing primers located in a region not affected by alternative splicing or single nucleotide polymorphisms was developed to study 104 DTC patients (13 of 104 with positive TgAb).Results: the assay is specific for thyroid tissue because we found mRNA Tg expression in normal thyroid tissue, but we did not find any mRNA Tg expression in any extrathyroidal tissues. Quantitative mRNA Tg levels were significantly different between patients free of disease (82 of 104) and those with metastases (22 of 104) (2.61 +/- 0.26 vs. 27.58 +/- 1.62 pg mRNA Tg/mu g RNA) (P < 0.0001). A cutoff point of 5.51 was able to discriminate between the two groups. in addition, the measurement of mRNA Tg was not affected by the presence of TgAb.Conclusion: This new mRNA Tg quantification is a reliable method that allowed us to differentiate patients free of disease from those with metastases, and it could represent an appropriate molecular marker for the follow-up of patients with DTC, especially those with positive TgAb. (J Clin Endocrinol Metab 95: 1726-1733, 2010)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Research-Fellowship GrantUniversidade Federal de São Paulo, Dept Med, Escola Paulista Med, Div Endocrinol,Lab Mol Endocrinol, BR-04039032 São Paulo, BrazilInst Israelita Ensino & Pesquisa Albert Einstein, Thyroid Dis Ctr, BR-05652000 São Paulo, BrazilFleury Med & Hlth, BR-01243001 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Escola Paulista Med, Div Endocrinol,Lab Mol Endocrinol, BR-04039032 São Paulo, BrazilFAPESP: 04/09934-7Research-Fellowship Grant: 05/55842-0Web of Scienc

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. in addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.Univ São Paulo, Fac Med, Inst Psiquiatria, Neurosci Lab,Dept Psiquiatria, BR-05403010 São Paulo, BrazilUniv São Paulo, Fac Med, Dept Bioquim, BR-05403010 São Paulo, BrazilUniv São Paulo, Fac Med, Lab Bioinformat, Inst Quim, BR-05403010 São Paulo, BrazilUniv São Paulo, Fac Med, Disciplina Oncol, Dept Radiol, BR-05403010 São Paulo, BrazilUniversidade Federal de São Paulo, Mol Endocrinol Lab, Dept Med & Morfol, São Paulo, BrazilHosp Canc AC Camargo, Dept Cirurg Cabeca & Pescoco & Otorrinolaringolog, São Paulo, SP, BrazilUniv Estadual Campinas, Inst Biol, Dept Genet & Evolucao, Lab Biol Mol & Genom Hemoctr, Campinas, SP, BrazilUniv Estadual Campinas, Inst Biol, Dept Genet & Evolucao, Lab Genom & Expressao, Campinas, SP, BrazilUniv Estadual Paulista, Dept Biol, Inst Biociencias, Araraquara, SP, BrazilFac Med Sao Jose Rio Preto, Dept Biol Mol, Sao Jose de Rio Preto, SP, BrazilUniv Estadual Paulista, Escola Farm, Dept Ciencias Biol, Araraquara, SP, BrazilUniversidade Federal de São Paulo, Mol Endocrinol Lab, Dept Med & Morfol, São Paulo, BrazilWeb of Scienc

A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms