Pico kit

Saliva samples (1 mL) were thawed on ice to avoid Extracellular Vesicles’ (EVs) thermal damage. Then, they were centrifuged at 4000× g for 30 min at 4 °C to remove any cell debris and aggregates. Supernatants were ultracentrifuged at 110,000× g for 75 min at 4 °C in order to pellet EVs, then stored at −20 °C.
miRNAs’ isolation from the obtained EVs was performed with the combination of the miRNeasy Kit and RNeasy Cleanup Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. They were eluted in 20 µL of nuclease-free water and stored at −80 °C until used. EV miRNAs’ quality and integrity were assessed through the “2100 Bioanalyzer RNA system” with the Pico Kit (Agilent Technologies, Santa Clara, CA, USA), but the concentration (ng/µL) was assessed by a Quantus Fluorometer (Promega, Milan, Italy).

Neurons from the CA1-CA3 regions of the hippocampus were collected by a PixCell IIe Laser Capture Microdissection Microscope (Life Technologies) from Sham, Severe TBI and Severe TBI + CM-treated rats as previously described in [28 (link)]. Cells were lysed in 100μl of lysis buffer and total RNA was isolated using RNAqueous Micro Kit (Ambion). Total RNA was assessed for quality and concentration on an Agilent Bioanalyzer (Agilent Technologies) using the Pico Kit (Agilent Technologies). 10 ng of total RNA was reverse transcribed and pre-amplified using the Qiagen RT2 Pre-amp cDNA synthesis kit along with RT2 PreAmp pathway primer mix specific to genes found in the Neurogenesis Array (Qiagen). QPCR was performed on a Roche Light Cycler 96 using the 96 well format. Data analysis was performed using the ΔΔCT method with Severe TBI and Severe TBI +CM compared relative to sham levels (n = 4/group) [S1 Fig displays genes found in Severe TBI+CM neurons to be significantly differentially expressed compared to Severe TBI alone]. PCR array profiling of laser captured neurons is described in detail in Boone et al., [29 (link)]. Bioinformatic and statistical analysis (principal component analysis and hierarchical clustering heatmap analysis) were performed with Qlucore Omics Explorer as previously described in Weisz et al., [30 (link)].