scholarly journals Validation of Reference Genes for Normalization Gene Expression in Reverse Transcription Quantitative PCR in Human Normal Thyroid and Goiter Tissue

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Raquel Weber ◽  
Ana Paula Santin Bertoni ◽  
Laura Walter Bessestil ◽  
Beatriz Maria de Azevedo Assis Brasil ◽  
llma Simoni Brum ◽  
...  

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate method for quantifying mRNA transcripts, but normalization of samples is a prerequisite for correct data interpretation. So, this study aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues. Beta-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); succinate dehydrogenase, subunit A, flavoprotein (Fp) (SDHA); hypoxanthine phosphoribosyltransferase I (HPRTI); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goiter tissues) by RT-qPCR. The mean Cq and the maximum fold change (MFC) and NormFinder software were used to assess the stability of the genes. As a result, ACTB gene was more stable than GAPDH, SDHA, HPRTI, YWHAZ, and B2M. In conclusion, ACTB could be used to normalize RT-qPCR data in normal thyroid and goiter tissues.

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Raquel Weber ◽  
Ana Paula Santin Bertoni ◽  
Laura Walter Bessestil ◽  
Ilma Simoni Brum ◽  
Tania Weber Furlanetto

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERαand ERβ) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n=16) and goiter (n=19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n=15) but in only 72% of goiter samples (n=13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.


2005 ◽  
Vol 19 (2) ◽  
pp. 79-87 ◽  
Author(s):  
G. Giubileo ◽  
F. Colao ◽  
A. Puiu ◽  
G. Panzironi ◽  
F. Brizzi ◽  
...  

An autofluorescence analysis has been performed on healthy as well as tumour thyroid tissue samples to distinguish follicular cancer from normal thyroid. Complete spectra and synchronous spectra have been recordered from properly stored samples. Fluorescence bands located at 350 nm and 400 nm has been observed in the analysed cancer samples.


2001 ◽  
Vol 356 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Phulwinder K. GROVER ◽  
Alan M. F. STAPLETON ◽  
Katsuhito MIYAZAWA ◽  
Rosemary Lyons RYALL

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or β-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to β-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean±S.D. of 0.37±0.07 (range 0.27–0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.


Author(s):  
Vladimir Zaichick

Background: Thyroid benign nodules (TBNs) are the most common diseases of this endocrine gland and are common worldwide. Among TBNs the colloid goiter (CG) and thyroid adenoma (TA) are very frequentdiseases. Evaluation of variant of TBNs is clinically important for subsequent therapeutic interventions, as well as for a clearer understanding the etiology of these disorders. The aim of this exploratory study was to examine differences in the content offifty trace elements (TE) in CG and TA tissues. Methods: Thyroid tissue levels of TE have prospectively evaluated in 46 patients with CG and 19 patients with TA. Measurements have performed using a combination of non-destructive and destructive methods: instrumental neutron activation analysis with high resolution spectrometry of long-lived radionuclides (INAA-LLR) and inductively coupled plasma mass spectrometry (ICPMS), respectively. Tissue samples were divided into two portions. One was used for morphological study while the other was intended for TE analysis. Results: It was observed that in both CG and TA tissues the contents of Ag, Al, Cr, Hg, Mn, Th, and Zn increased, whereas the levels of Au, Be, Cs, Pb, Rb, Sb, Sc, Th, Yb, and Zr were unchanged in comparison with normal thyroid tissue. No differences were found between the TE contents of CG and TA. Conclusions: From results obtained, it was possible to conclude that the common characteristics of CG and TA tissue samples were of a high level of Ag, Al, Cr, Hg, Mn, Th, and Zn in comparison with normal thyroid and, therefore, these TE could be involved in etiology and pathogenesis of thyroid disorders such as CG and TA.                    Peer Review History: Received: 12 November 2021; Revised: 15 December; Accepted: 31 December, Available online: 15 January 2022 Academic Editor: Ahmad Najib, Universitas Muslim Indonesia,  Indonesia, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.  Received file:                Reviewer's Comments: Average Peer review marks at initial stage: 6.5/10 Average Peer review marks at publication stage: 7.5/10 Reviewers: Prof. Dr. Hassan A.H. Al-Shamahy, Sana'a University, Yemen, [email protected] Rima Benatoui, Laboratory of Applied Neuroendocrinology, Department of Biology, Faculty of Science, Badji Mokhtar University Annaba, BP12 E L Hadjar–Algeria, [email protected] Similar Articles: COMPARISON OF LEVELS OF TWENTY CHEMICAL ELEMENTS IN NORMAL THYROID TISSUE AND HYPERTROPHIC THYROID TISSUE


2001 ◽  
pp. 591-597 ◽  
Author(s):  
D Russo ◽  
S Bulotta ◽  
R Bruno ◽  
F Arturi ◽  
P Giannasio ◽  
...  

OBJECTIVE: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). METHODS: The levels of NIS and pendrin proteins were analyzed in total protein extracts from nodular and non-nodular tissues by Western blot. RESULTS: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. CONCLUSIONS: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.


Author(s):  
VLADIMIR ZAICHICK

Objective: Colloid nodular goiter (CNG) is the most common disease of the thyroid, even in non-endemic regions, but the etiology of CNG is unclear. It is known that not merely iodine (I) but other chemical elements (ChE) are involved in goitrogenesis. The current study was performed to clarify the preferential accumulation of some ChE either in the colloid or in cells of the thyroid gland. Methods: Eight ChE: Bromine, calcium, chlorine (Cl), I, potassium, magnesium, manganese, and sodium (Na) in the thyroid tissues with diagnosed CNG were prospectively evaluated in 16 patients with macrofollicular CNG and 13 patients with microfollicular CNG. The control group included thyroid tissue samples from 105 healthy individuals. Measurements were conducted using non-destructive instrumental neutron activation analysis with high-resolution spectrometry of short-lived radionuclides. Results: It was found that in macrofollicular CNG, the mass fraction of Cl and Na was 2.57 and 1.82 times, respectively, higher than in tissues of the normal thyroid. In microfollicular CNG, the mass fraction of I was 59% lower, whereas the mass fraction of Na was 67% higher than in tissues of the normal thyroid. The level of I in macrofollicular goiter was 2.08 times higher than in microfollicular goiter Conclusion: There are substantial changes in ChE contents in the goitrous transformed tissue of the thyroid, which depend on the histology of the goiter.


2021 ◽  
Vol 12 (3) ◽  
pp. 098-107
Author(s):  
Vladimir Zaichick

Thyroid benign nodules (TBNs) are the most common lesions of this endocrine gland. Among TBNs the colloid goiter (CG), thyroiditis (T), and thyroid adenoma (TA) are the most frequent diseases. An evaluation of the variant of TBNs is clinically important for subsequent therapeutic interventions, as well as for more clear understanding the etiology of these disorders. The aim of this exploratory study was to examine differences in the content of bromine (Br), calcium (Ca), chlorine (Cl), iodine (I), potassium (K), magnesium (Mg), manganese (Mn), and sodium (Na) in tissues of CG, TA, and T. Thyroid tissue levels of eight chemical elements (ChE) were prospectively evaluated in 46 patients with CG, 19 patients with TA, and 12 patients with T. Measurements were performed using non-destructive instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides. Tissue samples were divided into two portions. One was used for morphological study while the other was intended for ChE analysis. It was observed that in CG, TA and T tissues content of Br was significantly higher, while level of I was lower than in normal thyroid tissue. I accumulation in T tissue was lower than in normal thyroid and CG. Abnormal increase in Br level and decrease in I level in all TBNs might demonstrate an involvement of these ChE in etiology and pathogenesis of TBNs. It was suosed that great losses of I in thyroid with T, in contract to little reduced levels of I content in thyroid with CG and TA, could possibly be explored for differencial diagnosis of T.


2013 ◽  
Vol 20 (4) ◽  
pp. 507-514 ◽  
Author(s):  
De-tao Yin ◽  
Wenxun Wu ◽  
Mingchuang Li ◽  
Qi-en Wang ◽  
Hongqiang Li ◽  
...  

The expression of the Dickkopf homolog 3 (DKK3) gene is downregulated in some human cancers, suggesting a possible tumor suppressor role of this gene. The role and regulation ofDKK3in thyroid cancer have not been examined. In this study, we explored the relationship of promoter methylation with the inactivation ofDKK3and tumor behaviors in papillary thyroid carcinoma (PTC). We used methylation-specific PCR and RT-PCR to examine the promoter methylation and expression ofDKK3and tumor characteristics. We found mRNA expression ofDKK3in 44.9% of the PTC tissue samples vs 100% of the matched normal thyroid tissue samples (P<0.01). In contrast, an opposite distribution pattern ofDKK3gene methylation was observed; specifically, 38.8% of the PTC tissue samples vs 0% of the matched normal thyroid tissue samples harboredDKK3methylation. An inverse correlation between the promoter methylation and mRNA expression ofDKK3in PTC tissue samples was also observed. Moreover, we also found an inverse correlation betweenDKK3expression and some aggressive pathological characteristics of PTC, including high TNM stages and lymph node metastasis, but a positive correlation betweenDKK3promoter hypermethylation and pathological aggressiveness of the tumor. Treatment of the PTC cell line TPC-1 with the demethylating agent 5-azaC reducedDKK3promoter methylation and enhanced its expression, establishing functionally the impact ofDKK3methylation on its expression. Our data thus for the first time demonstrate that theDKK3gene is a potential tumor suppressor gene in thyroid cancer and that aberrant promoter methylation is an important mechanism for its downregulation, which may play a role in the tumorigenesis and aggressiveness of PTC.


2001 ◽  
Vol 120 (5) ◽  
pp. A507-A507
Author(s):  
M BLAEKER ◽  
A WEERTH ◽  
L JONAS ◽  
M TOMETTEN ◽  
M SCHUTZ ◽  
...  

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