RNA Isolation from Serum Samples of Multiple Myeloma Patients

Venous blood samples were collected from previously untreated MM patients in serum separating tubes. The samples were processed within two hours of collection by centrifugation at 2400× g for 10 min, as described previously [38 (link)]. Serum samples were stored at −80 °C. Isolation of cell-free total RNA, including miRNA, was performed from serum using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Briefly, 200 µL of serum was mixed with Buffer RPL to ensure a complete lysis and to release and stabilize RNA from plasma proteins and extracellular vesicles. To allow for normalization of sample-to-sample variation in RNA isolation and to control the quality of RNA isolation, before purification, each serum sample was spiked with 22 nt synthetic miRNAs added to the RPL Buffer, using the RNA Spike-In Kit, For RT (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Briefly, each sample was spiked with UniSp2 (2 fmol), UniSp4 (0.02 fmol), UniSp5 (0.00002 fmol), each at 100-fold reductions in concentration. The sample was then mixed with Buffer RPP to precipitate proteins and inhibitors and then centrifuged. Isopropanol was added to the supernatant to provide the appropriate conditions for RNA molecules (>18 nucleotides) to bind to the silica membrane. The sample was then applied to a RNeasy UCP MinElute spin column, where RNA, including miRNA, binds to the membrane and other contaminants are washed away in subsequent wash steps. In the final step, total RNA (>18 nucleotides) was eluted using 20 µL of RNase-free water. The RNA quality was determined with Agilent High Sensitivity RNA ScreenTape using 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Only the samples with RIN > 7.0 were further processed. Directly after the isolation, RNA was subjected to reverse transcription.