Experion rna stdsens analysis kit

Cell lysates were centrifuged upon refreezing (12,000×g, 4 °C, 10 min) and chloroform was added to the supernatant (0.2 ml per 1 ml of TRI Reagent), mixed, and centrifuged after 5 min incubation at RT (12,000×g, 4 °C, 15 min). RNA-containing aqueous upper phase was collected and passed through gDNA Eliminator spin columns and then purified using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Isolated RNA was quantified by means of UV spectroscopy with NanoDrop 2000 (Thermo Scientific, Rockford, IL, USA), measured in duplicates, and its purity assessed by calculating ratios of absorbances at 260, 280, and 230 nm. RNA integrity was assessed using the Experion automated electrophoresis platform incorporating LabChip microfluidic technology and Experion RNA StdSens analysis kits (BioRad, Hercules, CA, USA). The RNA quality indicator (RQI) grading RNA from 10 (intact RNA) to 1 (degraded RNA) was calculated by Experion software for all samples. Possible presence of inhibitors in each RNA isolate was tested by calculating RT-qPCR reaction efficiencies from standard curves prepared by serial dilutions of respective cDNA samples (fivefold dilutions, 6 point-curve, conducted in duplicates).

Total RNA was isolated using phenol-chloroform extraction followed by purification with PureLink™ RNA Mini Kit (Thermo-Fisher Scientific, Waltham, MA, USA) involving on-column digestion of genomic DNA with PureLink™ DNase Set (Thermo-Fisher Scientific). Purified RNA isolates were quantified using NanoDrop 2000 (Thermo-Fisher Scientific). RNA purity was determined by calculating ratios of absorbance at 260, 280, and 230 nm. RNA integrity was evaluated using the Experion platform, incorporating LabChip microfluidic technology, and Experion RNA StdSens analysis kits (BioRad, Hercules, CA, USA). RNA quality indicator (RQI) score was calculated for each RNa sample and only RNA isolates with RQI ≥ 7, indicative of good RNA quality, were used for reversely-transcribed quantitative polymerize chain reaction (RT-qPCR).

Whole blood (3 mL) was collected into PAXgene Blood RNA Tubes and stored at −80 °C until RNA isolation. RNA was isolated using the complementary PAXgene Blood RNA Kit (Qiagen, Hilden, Germany) as instructed by the manufacturer. The concentration of isolated RNA was quantified using NanoDrop 2000 (ThermoScientific, Batavia, IL, USA) with the concomitant evaluation of RNA purity (ratios of absorbance at 260, 280, and 230 nm). RNA integrity was evaluated using the Experion platform incorporating LabChip microfluidic technology and Experion RNA StdSens analysis kits (BioRad, Hercules, CA, USA) and expressed as an RNA quality indicator (RQI) score with RQI = 1 indicative of degraded and RQI = 10 indicative of intact RNA. Only RNA isolates with RQI ≥ 7 were used for RT-qPCR. The possible presence of inhibitors in each RNA isolate was tested by calculating RT-qPCR efficiencies from standard curves prepared by serial dilutions of respective cDNA samples (five-fold dilutions, 6 point-curve, conducted in duplicates using SG qPCR Master Mix from EURx, Gdansk, Poland). A working dilution of cDNA 1:5 was found to effectively dilute reaction inhibitors and assure near 100% qPCR efficiencies.