Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma
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Molecular Medicine
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SHORT REPORT
Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma Daina Pamedytyte1* , Enrika Leipute1, Birute Zilaitiene3, Valdas Sarauskas2, Dalia Dauksiene3, Albertas Dauksa4 and Aurelija Zvirbliene1
Abstract Background: The 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, paraffinembedded (FFPE) PTC tissue age on estimated target miRNA expression. Methods: RQ-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. Results: The 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. Conclusions: It 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. Keywords: Stability, miRNA, Endogenous control, FFPE, Papillary thyroid carcinoma Background MicroRNAs (miRNAs) are small non-coding RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression (Bartel 2004). Disregulated miRNA expression has been reported in many human cancer cells including thyroid cancer. Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer and it was shown by many researchers that numerous miRNAs are aberrantly expressed during disease development or progression, making these *Correspondence: [email protected] 1 Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio Av. 7, 10257 Vilnius, Lithuania Full list of author information is available at the end of the article
miRNAs attractive as diagnostic or prognostic biomarkers (Leonardi et al. 2012). The ability to use specific miRNAs as biomarkers would assist in determining disease recurrence risk based on the miRNA expression in primary tumor and therefore would allow stratifying patients for a more personalized treatment. Currently the most popular method for miRNA quantitation is real-time quantitative PCR (RQ-PCR) due to its sensitivity and reproducibility. In order to get reliable data on relative miRNA expression by RQ-PCR, corrections must be made for variation between reactions introduced during all experimental steps from RNA purifica
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