Оптимизация методики определения числа копий TREC и KREC в цельной крови методом мультиплексной ПЦР в режиме реального времени
Аннотация
Работа посвящена оптимизации методики количественного определения числа копий TREC и KREC методом мультиплексной ПЦР с детекцией в режиме реального времени. Показано, что наиболее подходящим и точным методом определения ДНК является флюориметрический подход, т.к. распределение значений медианы для концентраций, измеренных на приборе Qubit, находится в районе единицы, что свидетельствует о хорошей сходимости результатов. Выяснено, что оптимальной температурой отжига праймеров является 60 °С, при которой наиболее эффективно нарабатываются продукты амплификации. Построены калибровочные кривые для определения числа копий TREC, KREC из расчета на 105 ядросодержащих клеток с использованием ПЦР-РВ. Отработанная методика была апробирована на образцах ДНК новорожденных, выделенных из цельной крови.
The work was devoted to the optimization of the method for quantitative determination of the number of copies of TREC and KREC by the method of multiplex real-time PCR (RQ-PCR). It was shown that the most suitable and accurate method for determining the DNA concentration is Qubit fluorometric assay, since the distribution of the median values for the concentrations measured by Qubit was within the region of unity, which indicated a good convergence of the results. It was found that the optimal annealing temperature for the developed RQ-PCR assay is 60°C, at which the most effective amplification were noted. Calibration curves were constructed to find the copy number of TREC, KREC in respect to 105 nucleated cells using RQ-PCR. The technique was tested on DNA samples of newborns isolated from whole blood.
The work was devoted to the optimization of the method for quantitative determination of the number of copies of TREC and KREC by the method of multiplex real-time PCR (RQ-PCR). It was shown that the most suitable and accurate method for determining the DNA concentration is Qubit fluorometric assay, since the distribution of the median values for the concentrations measured by Qubit was within the region of unity, which indicated a good convergence of the results. It was found that the optimal annealing temperature for the developed RQ-PCR assay is 60°C, at which the most effective amplification were noted. Calibration curves were constructed to find the copy number of TREC, KREC in respect to 105 nucleated cells using RQ-PCR. The technique was tested on DNA samples of newborns isolated from whole blood.