Practical approach to quantification of mRNA abundance using RT-qPCR, normalization of experimental data and MIQE

• Дата: 2016
• Автори: Obolenskaya, M.Yu., Kuklin, A.V., Rodrigez, R.R., Martsenyuk, O.P., Korneyeva, K.L., Docenko, V.A., Draguschenko, O.O.
• Формат: Стаття
• Мова: English
• Опубліковано: Інститут молекулярної біології і генетики НАН України 2016
• Назва видання: Вiopolymers and Cell
• Теми: Reviews
• Онлайн доступ: http://dspace.nbuv.gov.ua/handle/123456789/152828
• Назва журналу: Digital Library of Periodicals of National Academy of Sciences of Ukraine
• Цитувати: Practical approach to quantification of mRNA abundance using RT-qPCR, normalization of experimental data and MIQE / M.Yu. Obolenskaya, A.V. Kuklin, R.R. Rodrigez, O.P. Martsenyuk, K.L. Korneyeva, V.A. Docenko, O.O. Draguschenko // Вiopolymers and Cell. — 2016. — Т. 32, № 3. — С. 161-172. — Бібліогр.: 46 назв. — англ.

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) has become the most common method for characterizing expression patterns of individual mRNAs due to a large dynamic range of linear quantification, high speed, sensitivity, resolution and cost-effectiveness. However, the complexity of the protocol, variability of reagents, an inconsistent quality of biological samples, and the absence of standardized methods of data quantification may produce inconsistent results. In an effort to to standardize ithe procedure and assure high reliability of data, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines was defined and further extended by Prof. Bustin and colleagues (2004). These guidelines have been followed by the development of an XML-based real-time PCR data markup language (RDML) and a RDML data base for consistent reporting of RT-qPCR data created by the RDML consortium. Here we follow the RT-qPCR procedure step by step in compliance with MIQE requirements, local facilities and resources and our own experience in application of RT-qPCR methodology.