Abstract
BackgroundThe most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression.MethodsRQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years.ResultsThe stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time.ConclusionsIt is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.
Highlights
The most popular miRNA quantitation technique is real-time quantitative PCR (RQ-PCR) with relative gene expression method that requires an endogenous control (EC) gene for data normalization
The most popular endogenous controls used in papillary thyroid carcinoma (PTC) research As the first step in our research, we have analyzed the currently available published data on the ECs used for miRNA profiling in PTC studies
The levels of miRNA expression are different in tissue samples of different age we investigated how specific ECs can influence the results of the measured miRNA expression levels and evaluated whether the results of miRNA profiling depend on the FFPE PTC tissue sample age
Summary
The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. There are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). A thorough research on the most suitable ECs for miRNA expression normalizations in specific tissues is lacking. There are no published data on the most suitable ECs for miRNA expression normalization in thyroid tissue. Different ECs are used by different authors usually with little or no information on why the particular EC was chosen This can be one of the reasons why miRNA expression results in PTC differs substantially between the studies
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