Selection and validation of miRNAs as normalizers for profiling expression of microRNAs isolated from thyroid fine needle aspiration smears

Abstract

Fine needle aspiration cytology (FNAC) is currently the method of choice for malignancy prediction in thyroid nodules. Nevertheless, in some cases the interpretation of FNAC results may be problematic due to limitations of the method. The expression level of some microRNAs changes with the development of thyroid tumors, and its quantitation can be used to refine the FNAC results. For this quantitation to be reliable, the obtained data must be adequately normalized. Currently, no reference genes are universally recognized for quantitative assessments of microRNAs in thyroid nodules. The aim of the present study was the selection and validation of such reference genes. Expression of 800 microRNAs in 5 paired samples of thyroid surgical material corresponding to different histotypes of tumors was analyzed using NanoString technology and four of these (hsa-miR-151a-3p, -197-3p, -99a-5p and -214-3p) with the relatively low variation coefficient were selected. The possibility of use of the selected microRNAs and their combination as references was estimated by RT-qPCR on a sampling of cytological smears: benign (n=226), atypia of undetermined significance (n=9), suspicious for follicular neoplasm (n=61), suspicious for malignancy (n=19), medullary thyroid carcinoma (MTC) (n=32), papillary thyroid carcinoma (PTC) (n=54) and non-diagnostic material (ND) (n=34). In order to assess the expression stability of the references, geNorm algorithm was used. The maximum stability was observed for the normalization factor obtained by the combination of all 4 microRNAs. Further validation of the complex normalizer and individual selected microRNAs was performed using 5 different classification methods on 3 groups of FNAC smears from the analyzed batch: benign neoplasms, MTC and PTC. In all cases, the use of the complex classifier resulted in the reduced number of errors. On using the complex microRNA normalizer, the decision-tree method C4.5 makes it possible to distinguish between malignant and benign thyroid neoplasms in cytological smears with high overall accuracy (>91%).

Combined quantitation of HMGA2 mRNA, microRNAs, and mitochondrial-DNA content enables the identification and typing of thyroid tumors in fine-needle aspiration smears

“…The choice of the miRNA set was based on the analysis of published data and our original preliminary results. A list of 13 miRNAs – malignancy markers – was compiled (miR-144-5p, -145-5p, -155-5p, -146b-5p, -183-5p, -199b-5p, -221-3p, -223-3p, -31-5p, -375, -451a, -551b-3p, and -7-5p), for which a set of three reference miRNAs was selected too (miR-197-3p, -23a-3p, and -29b-3p) [9, 20–25].…”

Detection of high-grade neoplasia in air-dried cervical PAP smears by a microRNA-based classifier

“…From the material of cytological preparations, we succeeded in obtaining from 5 to 50 µg of total RNA of satisfactory quality (A 260/280: 1.5–1.8) and in sufficient concentration for quantitative measurements (120–550 ng/µl). The microRNA and U6 snRNA expression were analyzed by stem-loop qPCR as previously described ( 34 ). The list of microRNAs was made based on the meta-analysis of related literature data.…”

Analysis of Relative Expression of the Hmga2 Gene and Oncogenic Microrna-221 in Cytological Slides Obtained by a Fine-Needle Aspiration Biopsy of the Thyroid Nodules

“…Нормировку уровня миРНК-221 проводили на содержание референсной миРНК-197 [24] , где Ct -пороговый цикл реакции для соответствующей микроРНК.…”