For the study of RNA isolation technical parameters, ten lumbar
puncture-harvested CSF samples were mixed and spun at 3000 × g for 5 min.
Aliquots of 200 μl supernatant from the mixed sample were used for each
RNA isolation procedure. Four replicates were evaluated for each method. Total
RNA was extracted and compared using commercially available kits: MagMAX™
mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific),
miRCURY™ RNA Isolation Kit–Biofluids (Exiqon), miRNeasy
Serum/Plasma Kit (QIAGEN), NucleoSpin miRNA Plasma (MACHEREYNAGEL),
PureLink™ RNA Mini Kit (Thermo Fisher Scientific), and TRIzol LS reagent
(Thermo Fisher Scientific). RNA isolations were carried out following
manufacturers’ instructions except for TRIzol LS reagent which was done
exactly as described previously [3 (link), 22 (link), 23 (link)]. RNA isolation experiments were performed with or without
nucleic acid carriers, glycogen, and bacteriophage MS2 RNA (both from Roche).
RNA was eluted or dissolved with 30 μl of nuclease-free water containing
RNAsin (0.5 U/μl, Promega). After a preliminary comparison by single-tube
TaqMan® miRNA assays (data not shown), three kits including
miRCURY™ RNA Isolation Kit–Biofluids, miRNeasy Serum/Plasma Kit,
and TRIzol LS reagent were selected for further assessments by miR-15/107
TaqMan® Low-Density Array (TLDA) analysis [24 (link)].
puncture-harvested CSF samples were mixed and spun at 3000 × g for 5 min.
Aliquots of 200 μl supernatant from the mixed sample were used for each
RNA isolation procedure. Four replicates were evaluated for each method. Total
RNA was extracted and compared using commercially available kits: MagMAX™
mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific),
miRCURY™ RNA Isolation Kit–Biofluids (Exiqon), miRNeasy
Serum/Plasma Kit (QIAGEN), NucleoSpin miRNA Plasma (MACHEREYNAGEL),
PureLink™ RNA Mini Kit (Thermo Fisher Scientific), and TRIzol LS reagent
(Thermo Fisher Scientific). RNA isolations were carried out following
manufacturers’ instructions except for TRIzol LS reagent which was done
exactly as described previously [3 (link), 22 (link), 23 (link)]. RNA isolation experiments were performed with or without
nucleic acid carriers, glycogen, and bacteriophage MS2 RNA (both from Roche).
RNA was eluted or dissolved with 30 μl of nuclease-free water containing
RNAsin (0.5 U/μl, Promega). After a preliminary comparison by single-tube
TaqMan® miRNA assays (data not shown), three kits including
miRCURY™ RNA Isolation Kit–Biofluids, miRNeasy Serum/Plasma Kit,
and TRIzol LS reagent were selected for further assessments by miR-15/107
TaqMan® Low-Density Array (TLDA) analysis [24 (link)].
