animal: rat#5 Sex: male strain: Wistar tissue: brain brain region: hippocampus treatment: TBI activation agent: PBS treatment time: 7 days
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with RNeasy Mini Kit followed by DNase I treatment.
Label
SYBR Green
Label protocol
cDNA was synthesized with miScript II RT Kit (Qiagen, Hilden, Germany) using 250 ng of total RNA isolated from brain tissue samples as described in the previous section. Synthesized cDNA was quantified using miScript SYBR Green PCR Kit (Qiagen, Hillen, Germany) and a сustom miScript miRNA PCR Array (Cat. No./ID: 331231, Qiagen, Hilden, Germany) in the 96-well format for analysis of 45 miRNAs, SNORD61, SNORD72 (small nucleolar RNA, C/D box 61/72) in 2 samples per plate, and PPC (PCR positive control) offered by the manufacturer. The real-time PCR was carried out on a CFX96 system (BioRad, USA) according to the program: 95°С – 15 min, 45 cycles: 94°С – 15 s, 55°С – 30 s, 70°С – 30 s, followed by analysis of the melting curves of the obtained PCR-products plotted in the range of 55 °С – 95 °С.
Hybridization protocol
n/a
Scan protocol
n/a
Description
control SAMPLE 11
Data processing
For the normalization it uses the average of housekeeping genes: SNORD61, SNORD72 (small nucleolar RNA, C/D box 61/72). Target gene signals normalized to housekeeping genes; ΔСt = Ct(miRNA) – Ct(SNORD) Fold Change worksheet reports control/test ( all samples PBS treatment/ all samples EV treatment) ratios at 7 day for brain regions (cortex, striatum, hippocampus).
