Mircury spike in kit

Using a different RNA isolation kit, the miRNeasy Mini Kit (Qiagen, Germany), total RNA was extracted from the same serum samples (n = 39) following the manufacturer’s protocol. A synthetic RNA oligonucleotide mix obtained from the miRCURY Spike-In kit (Qiagen, Germany) was added to each sample at equimolar amounts prior to RNA extraction. These spike-ins were subsequently used to monitor RNA extraction efficiency. The extracted total RNA and miR were stored at –80°C until further analysis.
Further, from total RNA samples, cDNA was synthesized using the miRCURY RT Kit (Qiagen, Germany). Reaction conditions were set according to recommendations by the manufacturer and 2μL of total RNA was used as input in a 10μL reaction. Quantitative RT-PCR reactions were set up using miRCURY SYBR ^® green master mix with a 1:50 diluted cDNA and commercial LNA-enhanced primer assay. Reactions were performed in a 384-well primer spotted plates in a Roche LC480 II instrument (Roche, Germany), with the following temperature settings: 95°C for 10 min, 45 cycles of 95°C for 10 s, and 60°C for 60 s. Using the obtained Cq values, the relative miR expression was calculated, as aforementioned, normalizing against the geometric mean of an exogenous spike-in control (cel-miR-39-3p) and an endogenous control (hsa-miR-24-3p), as followed: ΔCq = Averaged Cq _- geomean –Average Cq _- miR.

Total RNA was extracted from 200 μL serum using the miRNeasy Mini Kit (Qiagen, Germany) as described by Kocijan et al (27 (link)). Briefly, precisely 200 μL of each serum sample were mixed with 1000 μL Qiazol and 1 μL of a mix of 3 synthetic spike-in controls (miRCURY spike-in kit, Qiagen, Cat No. 339390). Following a 10-minute incubation at room temperature, 200 μL chloroform were added to the lysates followed by centrifugation at 12 000g for 15 minutes at 4 °C. Exactly 650 μL of the upper aqueous phase were transferred to a miRNeasy mini column where RNA was precipitated with 750 μL ethanol followed by automated washing with RPE and RWT buffer in a QiaCube liquid handling robot. Finally, total RNA was eluted in 30 μL nuclease-free water and stored at −80 °C.