Total RNA (not enriched for small RNAs) was isolated from 0.5 mL CSF using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (ThermoFisher Scientific), with modifications [13 (link)]. RNA samples were concentrated (RNA Clean & Concentrator™−5 Kit; Zymo Research) and eluted into 8 µL of RNAse/DNase-free water. 3.2 µL of concentrated RNA was transcribed using MultiScribe™Reverse Transcriptase (ThermoFisher Scientific) in an 8 µL reaction volume, and 2.5 µL of the cDNA was pre-amplified in separate reactions with Megaplex™ RT Primer Pool Set v3.0.A or v3.0.B on a T-100 thermocycler (Bio-Rad, Hercules, CA) following the manufacturer’s instructions for detection of miRNAs with pre-amplification. The pre-amplification products were diluted into a prescribed final volume of 100 µL with water, and stored at −20°C.