Peripheral blood from each participant was collected and processed for serum extraction. Samples were centrifuged at 3000 g for 10 min at room temperature. Serum was transferred to new EP tubes and stored at − 80 °C for future analysis.
For microarray analysis, total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. miRNA labeling and hybridization were conducted with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA). For samples in the validation set, miRNAs were isolated from serum using the MiRNeasy Serum/Plasma kit (Qiagen, Valencia, CA, USA). RNA sample quality tests including RNA purity, total amount and integrity tests were performed. RNA purity was checked by the ratio of absorbance at 260 nm and 280 nm, a ratio of above 1.9 was accepted. The total RNA amount of each sample was at least 1 μg. RNA integrity was checked by agarose gel electrophoresis.
For microarray analysis, total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. miRNA labeling and hybridization were conducted with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA). For samples in the validation set, miRNAs were isolated from serum using the MiRNeasy Serum/Plasma kit (Qiagen, Valencia, CA, USA). RNA sample quality tests including RNA purity, total amount and integrity tests were performed. RNA purity was checked by the ratio of absorbance at 260 nm and 280 nm, a ratio of above 1.9 was accepted. The total RNA amount of each sample was at least 1 μg. RNA integrity was checked by agarose gel electrophoresis.
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