Total RNA was extracted from 0.5 mL of each CSF sample using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (ThermoFisher Scientific), modified to include two aqueous extractions during the organic phase extraction steps in order to maximize RNA recovery [13 (link)]. RNA samples were concentrated using the RNA Clean & Concentrator™-5 Kit (Zymo Research) and eluted in 9 µL RNAse/DNase-free water. RNA concentrations were initially measured on a set of test CSF samples using the Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). The average concentration for the test group was 133 pg/µL with a total RNA recovery of approximately 2 ng/mL CSF. The concentrated RNA samples were converted to cDNA and pre-amplified using a T-100 thermocycler (Bio-Rad, Hercules, CA) with Megaplex™ RT Primers, Human Pool Set v3.0, as per the manufacturer’s protocol (“Megaplex Pools For microRNA Expression Analysis”), following instructions for detection of miRNA with pre-amplification. The pre-amplification products were diluted into a prescribed final volume of 100 µL and stored at −20°C until ready for the final detection PCR reactions. Real-time PCR reactions followed the manufacturer’s protocol, using 18 µL of diluted pre-amplification product.