cardiovascularКардиоваскулярная терапия и профилактикаCardiovascular Therapy and Prevention1728-88002619-0125«SILICEA-POLIGRAF» LLC10.15829/1728-8800-2024-4121MNQAWTcardiovascular-4121Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLEАртефакты анализа при идентификации клеточных линий методом профилирования на основе коротких тандемных повторовArtifacts of analysis in cell line identification by short tandem repeat profilinghttps://orcid.org/0000-0003-2201-0472МальченковаА. А.MalchenkovaA. A.

Анастасия Алексеевна Мальченкова — м.н.с. молекулярно-генетической диагностики и персонализированной медицины НИИ экспериментальной диагностики и терапии опухолей.

Москва

Moscow

nastya.malchenkova@yandex.ruhttps://orcid.org/0000-0002-4660-8519КособоковаЕ. Н.KosobokovaE. N.

Екатерина Николаевна Кособокова — в.н.с., к.б.н. лаборатории молекулярно-генетической диагностики и персонализированной медицины НИИ экспериментальной диагностики и терапии опухолей.

Москва

Moscow

ekkos@mail.ruФГБУ "Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина" Минздрава РоссииBlokhin National Medical Research Center of Oncology2024301220242311Биобанкирование41214121Copyright © Мальченкова А.А., Кособокова Е.Н., 20252025Мальченкова А.А., Кособокова Е.Н.Malchenkova A.A., Kosobokova E.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://cardiovascular.elpub.ru/jour/article/view/4121Цель

Цель. Изучить и описать самые распространенные виды артефактов, которые возникают при детекции коротких тандемных повторов STR (short tandem repeat)-ампликонов методом капиллярного электрофореза и вызывают трудности в интерпретации полученных STR-профилей.

Материал и методы

Материал и методы. Клеточные линии получены из биоресурсной коллекции (БРК) клеточных линий ФГБУ "НМИЦ онкологии им. Н. Н. Блохина" Минздрава России. Выделение ДНК из суспензий клеток проводили по инструкциям производителей наборов DNeasy Blood & Tissue (QIAGEN, Германия) и ExtractDNA Blood & Cells (Евроген, Россия). Измерение концентрации ДНК осуществляли с помощью прибора Qubit 4.0 (Thermo Fisher Scientific, США) и набора реагентов Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, США). Мультиплексная полимеразная цепная реакция (ПЦР) была поставлена с помощью набора реактивов COrDIS "ЭКСПЕРТ26" (Гордиз, Россия). Капиллярный электрофорез продуктов ПЦР проводили на генетическом анализаторе Genetic Analyzer 3500xL (Applied Biosystems, США). Для обработки электрофореграмм использовали программу GeneMapper Software v6.0 (Thermo Fisher Scientific, США).

Результаты

Результаты. Изучены наиболее известные артефакты, связанные с методом STR-профилирования и последующим капиллярным электрофоретическим разделением ампликонов. Приведены случаи детектирования этих артефактов из личной практики. Даны рекомендации действий для улучшения картины электрофореза.

Заключение

Заключение. В работе изучены артефакты анализа при идентификации результатов STR-профилирования клеточных линий методом капиллярного электрофореза (STR-CE), с которыми исследователи сталкиваются в лабораторной практике. Подробно описаны и проиллюстрированы примерами из собственной практики распространенные виды артефактов анализа, которые вызывают трудности в интерпретации полученных при STR-профилировании результатов, а также возможные причины их появления. Приведены рекомендации по снижению количества неспецифических флуоресцентных сигналов и их интенсивности.

Aim

Aim. To study and describe the most common types of artifacts in detection of short tandem repeat (STR) amplicons by capillary electrophoresis and cause difficulties in interpreting the obtained STR profiles.

Material and methods

Material and methods. Cell lines were obtained from the bioresource collection of cell lines of the Blokhin National Medical Research Center of Oncology. DNA was isolated according to the manufacturer’s instructions of the DNeasy Blood & Tissue (QIAGEN, Germany) and ExtractDNA Blood & Cells (Evrogen, Russia) kits. DNA concentration was measured using a Qubit 4.0 device (Thermo Fisher Scientific, USA) and a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, USA). Multiplex PCR was performed using a COrDIS EXPERT26 reagent kit (Gordiz, Russia). Capillary electrophoresis of PCR products was performed on a 3500xL Genetic Analyzer (Applied Biosystems, USA). GeneMapper Software v6.0 (Thermo Fisher Scientific, USA) was used to process electrophoresis data.

Results

Results. The most well-known artifacts associated with the STR profiling and subsequent capillary electrophoretic separation of amplicons were studied. Cases of detection of these artifacts from personal practice are given. Recommendations for improving the electrophoresis pattern are given.

Conclusion

Conclusion. The paper studies the artifacts of analysis in cell line STR profiling by capillary electrophoresis (STR-CE), which researchers encounter in laboratory practice. Common types of analysis artifacts that cause difficulties in interpreting the results obtained during STR profiling, as well as possible reasons for their occurrence, are described in detail and illustrated with examples from our own practice. Recommendations are given for reducing the number of non-specific fluorescent signals and their intensity.

STR-профилированиеартефакты анализакапиллярный электрофорезSTR profilinganalysis artifactscapillary electrophoresisРабота выполнена при финансовой поддержке Министерства науки и высшего образования Российской Федерации в рамках научно-исследовательской работы № 075-15-2021-1060 "Создание и развитие биоресурсной коллекции генетически и фенотипически охарактеризованных клеточных линий и первичных опухолей человека"The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation within the research work № 075-15-2021-1060 "CreationReferencesNardone RM. Eradication of cross-contaminated cell lines: a call for action. Cell Biol Toxicol. 2007;23(6):367-72. doi:10.1007/s10565-007-9019-9.Nardone RM. Eradication of cross-contaminated cell lines: a call for action. Cell Biol Toxicol. 2007;23(6):367-72. doi:10.1007/s10565-007-9019-9.Freedman LP, Gibson MC, Ethier SP, et al. Reproducibility: changing the policies and culture of cell line authentication. 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The authors declare that there are no conflicts of interest present.