Abstract
A number of questions arise when choosing methods for experiments related to next-generation sequencing. On the one hand, while working with RNA extraction, added reagents and their residues can often inhibit sensitive chemicals with which the sequential synthesis is carried out for the sequencing. On the other hand, processing the same data using different software for the analysis can also impact on the sequencing results. This paper will present the step by step procedure for the preparation of samples taken from human biological fluids for subsequent sequencing of small RNAs, small noncoding RNAs in particular. Regarding the methods of extraction or isolation of RNAs, we found that low RNA yield can be improved significantly by following the isolation method for total RNA and its fractions included in Ambion’s MirVana PARIS kit, but only if using a special approach and modifying the organic extraction step. Compared to others, the methods supplied with commercially available kits at the time of researching this paper require only one organic extraction. This simple but, as it turned out, very useful modification makes it possible to access previously unavailable material. Potential advantages of this modification include a more complete profiling of small non-coding RNAs and a broader access to small sample volumes, as a rule, access to human biological fluids which can be prepared for RNA sequencing on the Illumina platform.
Highlights
Исследователи, которые хотят выполнить секвенирование нового поколения (NGS), должны решить ряд проблем, связанных со способами и методами подготовки образцов
Regarding the methods of extraction or isolation of RNAs, we found that low RNA yield can be improved significantly by following the isolation method for total RNA and its fractions included in Ambion’s MirVana PARIS kit, but only if using a special approach and modifying the organic extraction step
Potential advantages of this modification include a more complete profiling of small non-coding RNAs and a broader access to small sample volumes, as a rule, access to human biological fluids which can be prepared for RNA sequencing on the Illumina platform
Summary
Исследователи, которые хотят выполнить секвенирование нового поколения (NGS), должны решить ряд проблем, связанных со способами и методами подготовки образцов. Набор MirVana PARIS включает использование запатентованного лизирующего буфера с β-меркаптоэтанолом, который служит для денатурирования белков биологических жидкостей, экстракции РНК фенол-хлороформом из образца, с высоким содержанием белка, липидов и ДНК, с последующей очисткой на спиртовой/колоночной основе перед элюированием РНК. Хотя уровень для лучшей экстракции малых РНК с использованием модификаций, предложенных в этой работе, будет варьироваться в зависимости от конкретного набора, к которому применяется эта методика, было показано, что он обладает кроссплатформенной применимостью [10]. Инкубируем компоненты mirVana PARIS Kit при комнатной температуре
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