Qpcr kit

Total RNA from the liver homogenates was extracted using TRIzol solution, according to the manufacturer’s protocol (Invitrogen, Carlsbad, USA). A quantitative polymerase chain reaction (qPCR) kit (Vazyme, Nanjing, China) was used to quantify the gene expression level. The PCR conditions were as in a previous study [13 (link)]. The primer sequences were as follows: CsGRN, F-5′-CGC GGA TCC TGT AAA TAT AAC CAG ACT TG-3′, R-5′-TTA CTC GAG CGG AGC ACA GGT GTA GTG AT-3′; iNOS, F-5′-GCA CAG GAA ATG TTC ACC TAC-3′, R-5′-CAC GAT GGT GAC TTT GGC TAG-3′; Arg1, F-5′-ACG GAA GAA TCA GCC TGG TG-3′, R-5′-GTC CAC GTC TCT CAA GCC AA-3′; STAT3, F-5′-CAG CAG CTT GAC ACA CGG TA-3′, R-5′-AAA CAC CAA AGT GGC ATG TGA-3′; Bcl-2, F-5′-GGT GGG GTC ATG TGT GTG G-3′, R-5′-C GGT TCA GGT ACT CAG TCA TCC-3′; TFF3, F-5′-CCA AGC AAA CAA TCC AGA GCA-3′, R-5′-GCT CAG GAC TCG CTT CAT GG-3′; c-Myc, F-5′-GTC AGT TCG GGA AGG CTG TA-3′, R-5’-AAT CGG AGT TGG AAT CAG TCA C-3′; GAPDH, F-5′-ACG ACC ACT TTG TCA AGC TC-3′, R-5′-GTG AGG AGG GGA GAT TCA GT-3′.

To obtain RNA from each diaphragm tissue group, we used TRIzol reagent (Life Technologies, Carlsbad, CA) for isolation. The reverse transcription of the isolated RNA into cDNA was carried out using the qPCR kit (Vazyme, China). The experiments were performed according to the kit instructions, and the PCR reaction solution was prepared with a total system of 25 μL (Prolight Bio, China). To perform the PCR reaction, we set the temperature at 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Amplification specificity was verified by dissociating the product to generate a melting curve. As a reference gene, we utilised GAPDH. You can find the primer sequences for each gene listed in Table 1.

Total RNA was isolated from cultured cells using the RNAiso Plus reagent, and reverse transcription was carried out using HiScript II Q RT SuperMix from a qPCR kit (Vazyme). Real-time PCR was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme) on a real-time PCR system (Roche), with primers as listed in Supplemental Table 4. Expression levels of indicated genes were normalized to the internal control, and relative expression levels were evaluated using the 2^−ΔΔCT method. Each target was measured in triplicates.

Rats were anesthetized (urethane, 1.8 g/kg, i.p.) and brainstem was removed and sliced into coronal sections using a vibrotome (VT1200S; Leica Biosystems). The RVLM tissue was collected under a microscope based on the anatomical atlas. Total RNA was extracted using the RNA Extraction Kit (LS1040, Promega, China). After reverse transcription with HiScript III Super Mix, the qPCR was performed on a QuantStudio 6 real time PCR system (ABI, USA) using a qPCR kit (R323, Vazyme, China). The protocol was as follows: 95 °C for 30 s for 1 cycle; 95 °C for 10 s followed by 60 °C for 30 s for 40 cycles; 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s for 1 cycle. The primers were listed in Table 1. Each sample was analyzed in triplicate, and Gapdh was used as the internal control for normalization. The relative mRNA levels were calculated using the comparative Ct (2^−ΔΔCt) method and normalized by WKY rats.
Table 1

Primers sequences

Gene	Sense/antisense	Production size (bp)
Il1b	(+)5′-AATCTCACAGCAGCATCTCGACAAG-3′ (-)5′-TCCACGGGCAAGACATAGGTAGC-3′	98
Il6	(+)5′-ACTTCCAGCCAGTTGCCTTCTTG-3′	110
	(-)5′-TGGTCTGTTGTGGGTGGTATCCTC-3′
Tnf	(+)5′-ATGGGCTCCCTCTCATCAGTTCC-3′	111
	(-)5′-CCTCCGCTTGGTGGTTTGCTAC-3′
Gapdh	(+)5′-CTGCACCACCAACTGCTTAG-3′	119
	(-)5′-GGCCATCCACAGTCTTCTGA-3′

d-mannitol, d-sorbitol, d-arabitol, xylitol, ribitol, erythritol, glycerol, d-fructose-6-P, and d-fructose were obtained from Sigma-Aldrich (St. Louis, United States). Coomassie brilliant blue R-250, cofactors NADP, NAD, NADH, NADPH, and antibiotics were purchased from Sangon Biotech (Shanghai, China). New England Biolabs or Takara supplied all restriction enzymes. High-fidelity KOD-Fx, 2 × ready-to-use qTaq mixture for PCR were purchased from Toyobo (Japan). RNA extraction kit, reverse transcription kit, and qPCR kit were obtained from Vazyme Biotech Co., Ltd (Nanjing, China). All other chemicals were of analytical grade.

The genomic DNA of WT-PRV, rPRV-EGFP, and rPRV-ΔUL54-EGFP was extracted and used as the template for PCR kit (catalog no. AG11510; Agbio, Changsha, China) or qPCR kit (catalog no. Q311; Vazyme, Beijing, China). Total RNA was isolated from the infected or uninfected cells, and RT-qPCR kit (catalog no. A336; Genstar, Beijing, China) was used to quantify viral mRNA. The tests were performed according to the kit instructions. The primers for PCR, qPCR, and RT-qPCR are listed in Table 2.

After Gp. lemaneiformis RNA extracted (RNeasy Plant Mini Kit, OMEGA) and the first-strand cDNA synthesized (HiScript III RT SuperMix for qPCR, Vazyme), quantitative real-time PCR (qRT-PCR) reaction was carried out according to the qPCR kit instructions (Vazyme, Nanjing, China) to validate the expression of each gene during various stages of tetraspores releasing. The specific gene primers for quantitative real-time PCR (qRT-PCR) were designed with Primer Premier 5 (Tables S7 and S8). The qRT-PCR program was divided into three stages: Stage 1 was 95 °C for 30 s, Stage 2 was 95 °C for 10 s, 60 °C for 20 s, and cycle number was 40. Stage 3 was 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 10 s. In this experiment, per result consisted of four groups of biological repeats, each with four technical repetitions. Reference genes were 18S (18 s rRNA) and GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) [102 (link)], and the relative expression levels of the E3 genes were calculated by the methods of 2 ^−ΔΔCt [103 (link), 104 (link)].