Abstract
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.
Highlights
E specimen, method, experiment, and environment conditions
To determine the amplification efficiency, serial dilutions of several Complementary DNA (cDNA) templates were run for all 12 paired primers, and the results were used to depict a standard curve
In order to monitor changes occurring in the pattern of papillary thyroid carcinoma (PTC) gene expression for the biomarkers identification, confirming stable and reliable internal reference genes for relative Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is highly crucial
Summary
E specimen, method, experiment, and environment conditions. every specific experiment will require selecting the best reference gene to obtain accurate and reliable results[3]. The most important diagnostic method for thyroid cancers is fine-needle aspiration biopsy (FNAB), 15–30% of thyroid FNABs are cytologically indeterminate[6] For overcoming this problem, molecular based techniques are suggested to discover new molecular markers discriminating malignancy from benignity[7]. One of the most common methods for the investigation of thyroid tumor markers is qRT-PCR; there are no sufficient studies introducing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue samples. Without validation of reference genes, the results of gene expression studies in thyroid cancers are less reliable due to such unexpected behavior of HKGs. it is essential to determinate the genes with the highest level of expression stability for the improvement of relative qRT-PCR in further thyroid cancer research. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human PTC tissue samples
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