Mir 39 spike in control

Plasma samples were thawed at 37 °C and centrifuged at 1,900x g for 10 minutes to remove residual cells and debris7 (link). For each sample 500 μl of Qiazol (Qiagen) was added to 100 μl of plasma. Synthetic exogenous oligonucleotides were then added, as recommended by the manufacturer (Exiqon or Qiagen). For samples that underwent complete profiling in the discovery phase, synthetic UniSp2, UniSp4, and UniSp5 were added prior to RNA isolation to monitor for isolation efficiency, and UniSp6 and Cel-39 were added prior to reverse transcription (RT) to monitor RT-PCR efficiency (Exiqon). For samples studied in the validation phase, 5 μl of C. elegans miR-39 spike-in control obtained from Qiagen was added to monitor for technical variation. RNA was isolated using the miRNeasy Mini Kit according to the manufacturer’s protocol (Qiagen).

Blood was collected from mice via axillary vessels in 1.5 ml microcentrifuge tubes (Fisher), allowed to clot for 30 min at room temperature, and centrifuged at 900 × g for 10 min and 4°C. Serum layer was transferred to a new microcentrifuge tube and centrifuged for 10 min at 16,000 × g and 4°C, and the cleared supernatant was transferred to a new microcentrifuge tube without disturbing the pellet. One hundred microliter of serum sample per mouse was processed for RNA isolation using miRNeasy Serum/Plasma Kit (Qiagen) as per manufacturer's recommended protocol or stored at −80 till processing. Serum/Plasma C. elegans miR-39 Spike-In Control (Qiagen) was spiked into each sample prior to RNA purification as an internal control for miR expression profiling in serum to allow for monitoring of RNA recovery and purity, and reverse transcription efficiency. The RNA concentration and purity were determined by Qubit RNA assay broad range (Qubit RNA assay BR, Invitrogen) fluorometry. This reagent specifically binds to RNA only and does not detect DNA, protein or free nucleotides. Additionally, spectrophotometric analysis of all samples using Epoch Gen 5 spectrophotometer (Biotek) showed that all RNA samples had A260/280 ratios ≥1.8.