RNA is extracted from whole blood using the PAXgene™ Blood RNA System Kit (Qiagen, Düsseldorf, Germany) employing an amended version of the manufacturer’s guidelines. Briefly, the samples are removed from −80 °C and incubated at room temperature for 2 h to ensure complete lysis. Following lysis, the tubes are centrifuged for 10 min at 5000× g (Boseo M-24 centrifuge, Hamburg, Germany), the supernatant is decanted, and 500 μL of RNase-free water is added to the pellet. The tube is vortexed to thoroughly resuspend the pellet, centrifuged for 10 min at 5000× g, and the entire supernatant discarded. The remaining pelleted lysate is re-suspended in 360 μL of buffer BR1 by vortexing, and the manufacturer’s protocol is followed from this step [14 (link),15 ].
Freshly extracted RNA is measured using a NanoDrop ND-1000 UV-visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). The software displays the concentration in ng/μL. There is also a quality output, which provides 260/280 and 260/230 ratios enabling purity estimations. RNA integrity is additionally assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and poor quality samples are excluded from the analysis [15 ].
Freshly extracted RNA is measured using a NanoDrop ND-1000 UV-visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). The software displays the concentration in ng/μL. There is also a quality output, which provides 260/280 and 260/230 ratios enabling purity estimations. RNA integrity is additionally assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and poor quality samples are excluded from the analysis [15 ].
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