Abi 7500 fast qpcr system

The recombinant plasmid standard was serially diluted to a range from 5 to 5×10⁶ copies per microliter. Two µl of each dilution were utilized for qPCR assay on an ABI 7500 Fast qPCR system with 2.0.1 version software (Applied Biosystems, USA). The reaction mix (20 µl) contained 2 µl genomic DNA, 0.6 µM of primers VP28–140F and VP28–140R (Table 1), and 10 µl Maxima SYBR Green qPCR Master Mix (2×) (Fermentas, MBI). The thermal cycle program was described as follows: 95°C for 10 min, followed by 40 cycles of 94°C for 15 s, 60°C for 60 s.

All clinical samples and plasmid standards were run in triplicate following manufacturer recommendation of the ABI7500 Fast qPCR System (Applied Biosystems, Inc Foster, CA). The qPCR was performed according to the conditions (Table 1). Quantification of each target gene copy numbers was obtained by comparison with standard curves.

Total RNA was extracted from cells using Trizol (15596-026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quantity and quality were measured by NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNAs (cDNAs) were synthesized using 0.5 mg of total RNA, oligo(dT)12–18 primers, and a ReverTra Ace qPCR RT kit (FSQ-101, Toyobo, Osaka, Japan) following the manufacturer’s protocol. Gene expression was detected by qPCR using the cDNAs, THUNDERBIRD SYBR qPCR mix reagents (QPS-201, Toyobo, Osaka, Japan), and gene-specific oligonucleotide primers (listed in Supplementary Table S2) with an ABI 7500 fast qPCR system (Applied Biosystems, Carlsbad, CA, USA). The expression level of β-actin was used to normalize the relative abundance of RNAs. Significance was determined by taking the average of the β-actin-normalized 2^−ΔΔCT values.

Total RNA was extracted from bacterial cultures by the hot acid-phenol method as previously described (Bouffartigues et al., 2012 (link)) and tested by PCR for the absence of contaminating DNA using the primers pair FcfrX/RcfrX (Supplementary Table S4). The RNA concentration, the protein and solvent contaminations were determined by measuring the absorbance at 260, 280, and 230 nm, respectively. Total RNA quality was checked on a 2% agarose gel/1X-TAE electrophoresis buffer prior to use for cDNA synthesis. The mRNAs of interest were quantified by real-time PCR amplification of their cDNAs. Each primers pair was validated by verifying that the PCR efficiency was above 0.90, and that a single PCR product with the expected Tm was obtained. PCR were performed on cDNAs at least in triplicate and carried out in SYBR Green PCR Master Mix^TM (Applied Biosystems), with 300 nM of each primer (ABI 7500 Fast Q-PCR system, Applied Biosystems). The cDNA-generated signals were internally corrected with the 16S rRNA cDNA signal. The mRNA expression level was calculated by comparing threshold cycle (Ct) of target genes with control sample group and the relative quantification (RQ) data was determined with the 2^–ΔΔCt method using the RQ manager v1.2.1 and the Microsoft-excel based softwares (Muller et al., 2002 (link)).

Total RNA from the spinal dorsal horn of the ipsilateral or contralateral L4–L6 segments washed by RNase-Free water was isolated using Trizol (Invitrogen, USA) according to the manufacturer’s instruction. The RNA was then quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA). The cDNA was synthesized using a PrimeScript^TM RT reagent Kit with gRNA (Takara, Japan). Gene level was evaluated using the SYBR green PCR master mix (Thermo Fisher Scientific, USA) in an ABI 7500 Fast qPCR system (Applied Biosystems, USA) according to manufacture instruction. Primers of targeting genes were synthesized (TREM2 (sense primer: 5′-GGA ACC GTC ACC ATC ACT CT-3′, antisense primer: 5′-ATG CTG GCT GCA AGA AAC TT-3′); TNF-α (sense primer: 5′-GTG GAA CTG GCA GAA GAG GC-3′, antisense primer: 5′-AGA CAG AAG AGC GTG GTG GC-3′); IL-1β (sense primer: 5′-CTG TGT CTT TCC CGT GGA CC-3′, antisense primer: 5′-CAG CTC ATA TGG GTC CGA CA-3′); IL-6 (sense primer: 5′-TTC CAT CCA GTT GCC TTC TT-3′, antisense primer: 5′-CAG AAT TGC CAT TGC ACA AC-3′); GAPDH (sense primer: 5′-AGT GTT TCC TCG TCC CGT AGA-3′, antisense primer: 5′-TTG CCG TGA GTG GAG TCA TAC-3′)). The GAPDH, a housekeeping gene, was used as control. Results were normalized to GAPDH and examined using the comparative CT method.

Total RNA was extracted from 80%‐confluent cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on‐column DNase digestion (RNase‐Free DNase Set, Qiagen, Hilden, Germany). 4 μg of RNA was then used for cDNA preparation using the High‐Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real‐time PCR was performed using TaqMan Universal Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) with primer‐appropriate Universal ProbeLibrary probes (Roche, Basel, Switzerland) and the ABI 7500 FAST qPCR system (Thermo Fisher Scientific, Waltham, MA, USA). CASC3 gene was used as a normalising control. Gene expression is shown relative to control cell levels. Primers (Thermo Fisher Scientific, Waltham, MA, USA) used in this study are provided in Table EV6.

RNA was extracted from cells (70–80% confluence) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion (RNase-Free DNase Set, Qiagen, Hilden, Germany). cDNA was prepared from 4 µg RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using TaqMan Universal Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) with primer-appropriate Universal ProbeLibrary probes (Roche, Basel, Switzerland) and the ABI 7500 FAST qPCR system (Thermo Fisher Scientific, Waltham, MA, USA). Gene expression was normalised to the reference CASC3 gene, and is shown relative to levels in control cells. A list of the primers (Thermo Fisher Scientific, Waltham, MA, USA) used in this study is provided in Supplementary Table 1.