Selection and validation of miRNAs as normalizers for profiling expression of microRNAs isolated from thyroid fine needle aspiration smears.
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%).
Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Sergei E
SE
Titov
The Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Pavel S
PS
Demenkov
Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Mikhail K
MK
Ivanov
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Ekaterina S
ES
Malakhina
Non-governmental Healthcare Institution 'Railroad Clinical Hospital on the Station Novosibirsk-Glavny', Joint Stock Company 'Russian Railways', Novosibirsk 630003, Russia.
Tatiana L
TL
Poloz
AO Vector-Best, Novosibirsk 630117, Russia.
Elena V
EV
Tsivlikova
AO Vector-Best, Novosibirsk 630117, Russia.
Maria S
MS
Ganzha
Novosibirsk Municipal Budgetary Healthcare Institution 'Municipal Clinical Hospital #1', Novosibirsk 630047, Russia.
Sergei P
SP
Shevchenko
Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk 630117, Russia.
Lyudmila F
LF
Gulyaeva
Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Nikolay N
NN
Kolesnikov
D014408:Biomarkers, Tumor / Q000096:biosynthesis* / Q000235:genetics; D044963:Biopsy, Fine-Needle; D002277:Carcinoma / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D018278:Carcinoma, Neuroendocrine / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D002291:Carcinoma, Papillary; D003581:Cytodiagnosis / Q000379:methods; D003937:Diagnosis, Differential; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D000077273:Thyroid Cancer, Papillary; D013961:Thyroid Gland / Q000378:metabolism / Q000473:pathology; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology
D014408:Biomarkers, Tumor; D035683:MicroRNAs
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