Taqman universal master mix 2

Trizol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) was applied to extract total RNA from cells. The expression levels of LINC00094 and GAPDH were detected by One‐Step SYBR PrimeScript RT‐PCR Kit (Perfect Real Time; Takara Bio, Inc., Kusatsu, Japan). The expression levels of miR‐224‐5p/miR‐497‐5p and U6 were detected by TaqMan miRNA Reverse Transcription kit and Taqman Universal Master Mix II (Applied Biosystems, Foster City, CA, USA). High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and Taqman Universal Master Mix II were used to perform Endophilin‐1 and GAPDH expression. The relative quantification (2^−△△Ct) method was performed to normalize and calculate relative gene expression values. Primers and probes were listed in Table S1.

The viral DNA was extracted with phenol/chloroform solution and precipitated from the organic phase. The procedures were published previously [32 (link)]. The DNA pellet was washed twice in a solution containing 0.1 M trisodium citrate in 10% ethanol and dissolved in 8 mM NaOH. The DNA concentration was determined by fluorometer analysis with the Qubit double-stranded DNA (dsDNA) HS (High Sensitivity) Assay Kit according to the manufacturer’s instructions. Viral DNA amplification was carried out using TaqMan™ Universal Master Mix II (Applied Biosystems™, Foster City, CA, USA) in a 50 µL reaction mixture containing TaqMan Universal Master Mix II, DNA (100 ng), HSV-1 forward (10 µM), and HSV-1 reverse (10 µM) primers, TaqMan probe (5 µM). The sequence of primers is shown in Table 2. The amplification was carried out on Applied Biosystems 7300 Real-Time PCR System under the following conditions: 10 min at 95 °C, 60 s at 95 °C for 40 cycles, 30 s at 60 °C, and 30 s at 72 °C. Absolute quantification Real-time PCR using a specific TaqMan probe was performed to detect viral DNA. Viral load was derived from the threshold cycle (CT) using the standard curve generated in parallel, and the result is expressed as a concentration in µg of DNA/µL.

Blood samples were obtained from all subjects, and genomic DNA was isolated with the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The extracted DNA was eluted in TE buffer and stored at - 80 °C for further use. Two SNPs of PD-L1 gene, including rs2297136 (A > G) and rs4143815 (C > G), were examined by the real-time polymerase chain reaction (real-time PCR). The SNPs selection was based on those previously evaluated in relation to cancer or those with evidence of functional significance. The PCR mixture was prepared for each sample by using 10 μL TaqMan™ Universal Master Mix II (Thermo Fisher Scientific, USA), 0.5 μL of TaqMan™ SNP Genotyping Assay (40X) (ready-made assay, Cat. No: 4351379, Thermo Fisher Scientific, USA), 1 μL genomic DNA, and 8.5 μL RNase-free water to reach a total volume of 20 μL. A negative control consisting of 10 μL TaqMan™ Universal Master Mix II, 0.5 μL of TaqMan™ SNP Genotyping Assay (40X), and 9.5 μL RNase-free water was also prepared for each SNP. The real-time PCR cycling was initiated with UNG incubation at 60 °C for 2 min, polymerase activation at 95 °C for 10 min, followed by 40 cycles of (denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min).

Total RNA was extracted from CA1 region of hippocampal (Fig. 1b), or cultured HT22 cells (Figs. 1d, 2a, 3a, 4a, 4b, 5a, 6a and 7A) by using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA). RNA concentration and quality of each sample were determined with Nanodrop Spectrophotometer (ND-100) by the 260/280 nm ratio. The primers for Snhg8, Hoxa13, and β-actin were synthesized from Takara Bio Inc. The expression levels of miR-384 and U6 (Applied Biosystems, Foster City, CA, USA) were examined with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) and Taqman Universal Master Mix II (Life Technologies Corporation, Carlsbad, CA, USA).
Snhg8: forward 5′-GACACAAGGTGGCTATGGTGCTG-3′,
reverse 5′-CATGGTGGTCGTCGCGCTAAC-3′;
Hoxa13: forward5′-TACTTCGGCAGCGGCTACTACC-3′,
reverse 5′-CGGCGGTGTCCATGTACTTGTC-3′;
FAM3A: forward 5′-TTGGCCTTCCTCGAATTCAGCAG-3′,
reverse 5′-GCTCAGGTGCTCTTCAGGACAAG-3′;
β-actin: forward 5′-GTGACGTTGACATCCGTAAAGA-3′,
reverse 5′-GTAACAGTCCGCCTAGAAGCAC-3′.
The expression levels of miR-384 and U6 (Applied Biosystems, Foster City, CA, USA) were examined with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA) and Taqman UniversalMaster Mix II (Life Technologies Corporation, Carlsbad, CA, USA). The relative quantification 2^−△△Ct method was applied to calculate the gene expression.

Tissues were collected at appropriate time points and snap-frozen in liquid nitrogen. Total RNA was isolated using a Qiagen RNeasy Plus Isolation Kit followed by cDNA synthesis (High Capacity cDNA RT kit, Applied Biosystems, 4374966). RT-PCR was performed using TaqMan Universal Master Mix II (Fisher, 4440038), according to manufacturer’s protocol. Primers used were: Myc (Fisher, Mm00487804_m1) and Tbp (Fisher, Mm00446973_m1). Samples were analysed in triplicate on an Eppendorf Mastercycler Realpex 2 with with Eppendorf Quantstudio design & Statistical Analysis Vl.2 Software. Tbp was used as an internal amplification control.

Reverse transcription was performed with the TaqMan™ microRNA RT kit and rodent Megaplex RT primer pools set v3.0 (Fisher Scientific, Illkirch, France) in 7.5 µL reaction volume, following manufacturer’s instructions, on a Mastercycler® X50 (Eppendorf, Hamburg, Germany). The resulting cDNAs (2.5 µL) were pre-amplified (12 cycles) using Megaplex PreAmp primers and TaqMan PreAmp mastermix (Fisher Scientific) in a final volume of 25 µL, on the Mastercycler X50 (Eppendorf). The PreAmp products were then diluted to 100 µL with TE buffer, and 9 µL of diluted PreAmp products were used to prepare 900 µL of PCR reaction mix with TaqMan Universal Mastermix II, no UNG (Fisher Scientific), to load each of the 2 TaqMan low density array (TLDA) Rodent MicroRNA A + B Cards Set v3.0 (Fisher Scientific). Quantitative (q) PCR was run on an ABI 7900HT instrument (Applied Biosystems, Foster City, CA) for 40 cycles. Cycle thresholds (Ct) for each assay were determined using automatic baseline with Expression-Suite Software v1.3 (Applied Biosystems), and Ct values > 37 were discarded. Global normalization was applied to this dataset using DataAssist Software v3.01 (Applied Biosystems) to compute Delta-Ct values for subsequent multivariate analyses.