Hipure universal rna kit

To detect the mRNA levels of TREM2, FoxO3a and inflammatory cytokines (eg IL-1β, IL-6, TNF-α), the total RNA was extracted from hippocampus tissue and cells using Hi-pure universal RNA kit (Magen) according to the manufacturer's protocol. The concentration of mRNA was determined by Nanodrop 2000 (Thermo Fisher Scientific). The cDNA was synthesized using iScript cDNA Synthesis kit (Bio-Rad), and the reaction of qRT-PCR was performed by GoScript^TMqPCR Marster Mix (Promega, USA). Targeting genes were amplified by the following primers: TREM2 (sense primer: 5′-CTG GAA CCG TCA CCA TCA CT-3′, antisense primer: 5′-CAC CCT CGA AAC TCG ATG AC-3′); FoxO3a (sense primer: 5′-GAG TGA CTC CAG CAG CCT TG-3′, antisense primer: 5′-ATT CCA AGC TCC CAT TGA AC-3′); TNF-α (sense primer: 5′-TCA CTG GAG CCT CGA ATG TC-3′, antisense primer: 5′-TCT GTG AGG AAG GCT GTG CA-3′); IL-1β (sense primer:5′-TGT GTA ATG AAA GAC GGC ACA C-3′, antisense primer: 5′-CTT GTG AGG TGC TGA TGT ACC A-3′); IL-6 (sense primer: 5′-CCA CGG CCT TCC CTA CTT C-3′, antisense primer: 5′-TTG GGA GTG GTA TCC TCT GTG A -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 gene of GAPDH was considered as housekeeping gene and the expression of the interested genes were all normalized to GAPDH. The data was analyzed using the comparative CT method.

The strains were cultured in liquid CM for 4 days at a speed of 110 rpm. The mycelia were filtered, washed with sterilized ddH₂O, dried with absorbent paper, and further dry frozen in liquid nitrogen. Total RNA was extracted from the individual strains using a HiPure Universal RNA kit (R4130-02; Magen, China). The expression of MoOeIF3K under different stress conditions and expression of individual subunits of the MoeIF3 complex in ΔMoOeif3k were monitored by quantitative real-time PCR (qRT-PCR) assays. Reverse transcription of RNAs was performed using the PrimeScript RT regent Kit with gDNA Eraser (RR047A; Takara, Japan). A 10-μl reaction mix was formulated as follows: 5 μl TB green, 3.4 μl RNase free water, 0.3 μl of each 10 μM forward and reverse primers listed in Supplementary Table 1 and 1 μl cDNA template. qRT-PCR was carried out with Eppendorf Realplex2 master cycler (AG 223341; Eppendorf, Hamburg, Germany). The raw qRT-PCR data were analyzed using the formula delta delta-CT (2^–ΔΔCT) method described by Rao et al. (2013) and Abdul et al. (2018) (link). The expression level of tubulin was used as the reference or internal control. Error bars represent mean ± SD. The data were obtained from three independent biological experiments with three technical replicates for each independent experiment.

To examine the transcription level of N, P, M, G, and L mRNAs, the viral genomic RNA (vRNA) level and leader RNA (LeRNA) level, the monolayers of NA cells were infected with rHEP-Flury and rescued viruses, respectively, at a multiplicity of infection (MOI) of 3 to make sure every cell was infected. The cells were washed with phosphate buffered saline (PBS) and harvested after 12 h incubation at 34 °C, then the total RNA was purified using a HiPure Universal RNA kit (Magen, Guangzhou, China) and reverse transcription (RT) was carried out using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. The transcription level of each gene was determined, as previously described [17 (link)]. Table S1 provides primer sequence details. A SYBR Green Master Mix (Vazyme Biotech CO., Ltd., Nanjing, China) was used for the real-time PCR (qPCR) and qPCR was carried out in a CFX384 Real-Time System (Bio-Rad, Hercules, CA, USA). The copy numbers of each target gene were normalized to the housekeeping gene beta actin (β-actin).

Total RNA was respectively isolated from frozen BF samples collected from nine female pigeon squabs across the three developmental stages (three biological replicates each) by HiPure Universal RNA Kit (Magen, Guangzhou, China) according to the manufacturer’s protocol. The integrity of total RNA was evaluated using an Agilent 2100 Bioanalyzer. The RNA concentrations were assessed with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Massachusetts, USA). Each total RNA sample was first treated to remove ribosomal RNA using the Ribo-Zero™ kit (Epicentre,Madison, WI, USA). The rRNA-depleted RNA was used to prepare the stranded-specific RNA-seq library following the manufacturer’s standard procedures. Subsequently, prepared cDNA libraries were sequenced by an Illumina NovaSeq 6000 platform and the paired-end reads were obtained. All the RNA-seq data have been deposited in NCBI database with accession number GSE183791.

To evaluate RABV load at the inoculation site, KM mice (6–7 weeks of age) were inoculated in the right hind leg with 1.0 × 10⁵FFU rGDSH-G349 or GD-SH-01. After the mice were humanely killed, the right hind leg muscles of five mice from each group were removed from infected mice at 1, 3, 5, 7, and 9 dpi and grinded in liquid nitrogen and then lysed in Magzol reagent (Magen). Total RNA was extracted using the HiPure Universal RNA Kit (Magen), and qRT-PCR was performed as described previously (Wang et al., 2014 (link)) to determine RABV genomic RNA in muscles using primers amplifying leader RNA and partial N. Genomic RNA was normalized to GAPDH.