Qiaexpert spectrophotometer

According to the manufacturer's manufacturer, total RNA, including small RNAs, was isolated and purified from the buffy coat of patients using miReasy Mini Kit (Cat #217004; Qiagen, Hilden, Germany) instruction for cells samples. The RNA concentration and purity were detected using a QIAexpert spectrophotometer (Cat #1038703; Qiagen). Then complementary DNA (cDNA) was synthesized from 10 ng of total RNA using a TaqMan MicroRNA Reverse Transcription Kit (Cat #4366597; Applied Biosystem Lithuania) with a specific RT primer (Cat #4440887 hsa-miR21-5p; Cat #4427975 U6 snRNA; Applied Biosystem, Lithuania) according to the manufacturer's protocol. miRNA expression was measured with a Rotor-Gene Q (Cat #R0515102; Qiagen) using PrimeTime Gene Expression Master Mix (Cat #1055772; IDT) and the TM primer (Cat #4440887 hsa-miR21-5p; Cat #4427975 U6 snRNA Applied Biosystem). The two-step PCR condition was 3 m at 95°C, 45 cycles with 5 s at 95°C, and 30 s at 60°C (10 μl reaction volume and 2 μl cDNA template). All samples were run in triplicate. A threshold cycle (C_t) value was obtained for each amplification cycle, and ΔC_t was calculated as the C_t difference between target miRNA and U6. miRNA expression was calculated using the 2^−ΔΔCt method.

Fresh brains were collected for qPCR to evaluate inflammatory gene expression. The cortex was processed for RNA isolation using the RNeasy mini kit (Qiagen, Hilden, Germany) and a QIAcube (Qiagen), as per the manufacturer’s protocol. Eluted RNA concentration and purity were determined using a Qiagen QIAexpert spectrophotometer. One microgram of eluted RNA from each sample was reversed transcribed into cDNA using a QuantiTect Reverse Transcription kit (Qiagen) and diluted to 1:10.
Samples were pipetted into a 192-well plate using a Qiagility liquid handling robot (Qiagen). Samples were run in duplicate, and cDNA was amplified with a QuantStudio 7 Flex Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). TaqMan fast advanced gene expression assays (Thermo Fisher Scientific) were used for quantification of gene expression, as outlined in Supplementary Table S3.
Relative gene expression ratios were calculated using the 2^−ΔΔCT method and normalized to the geometric mean of the housekeeping genes (Ppia and Hprt), which we have previously shown to be stable in the brains of C57BL/6 mice [47 (link)]. The expression of each gene was normalized to control mice.

The THP-1 acute monocytic leukemic cell line (ATCC: The Global Bioresource Center) was expanded in complete RPMI medium composed of 10% Fetal Bovine Serum, supplemented with 1% Penicillin-Streptomycin (all from Merck KGaA) with the addition of L-glutamine, sodium pyruvate and 2-mercaptoethanol (1.7 mM, 0.87 mM and 0.02 mM, respectively). Cells were plated in a 6-well plate in complete RPMI medium and stimulated with 20 ng/ml of interferon-gamma (IFNγ) and/or 5 ng/ml lipopolysaccharide (LPS) for 24 hours. Following stimulation, cells were centrifuged at 350 g for 5 min, lysed in the RLT plus buffer (Qiagen) and kept at -80C until further use. Genomic DNA was extracted using the DNA/RNA Mini Kit (Qiagen) according to manufacturer’s instructions. The DNA was eluted in Elution Buffer (10 mM Tris-Cl, pH 8.5) and the DNA concentration was measured using the Qubit dsDNA Broad Range kit (Thermo Fischer Scientific). The purity of the extracted DNA was verified using the QIAexpert spectrophotometer (Qiagen), considering the OD260/280 absorbance ratio.