Rna pre pure kit

Macrophages were infected with the S. pneumoniae D39 at an MOI of 1 for 9 h. Total RNA was extracted by using the RNA Pre-Pure kit (Tiangen, Beijing, China). Complementary DNA was then synthesized by PrimeScript RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was carried out using SsoFast Eva Green Super-Mix (Bio-Rad, Hercules, CA, USA) and performed on a Bio-Rad CFX 96 instrument. Primers were used as follows:
IL-1β forward 5′- GAA ATG CCA CCT TTT GAC AGT G and reverse 5′- TGG ATG CTC TCA TCA GGA CAG, β-actin forward 5′-TGG AAT CCT GTG GCA TCC ATG AAA C, and reverse 5′- TAA AAC GCA GCT CAG TAA CAG TCC G.

Mice and macrophages were infected with P. multocida as described above. The samples (BALF and lung tissue) were collected at 3, 6, 9, and 24 h after infection and total RNA (500 ng) was extracted using the RNA Pre-Pure kit (Tiangen, Beijing, China). Then, complementary DNA was synthesized with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative real-time RT PCR was carried out using SsoFast Eva Green Super-Mix (Bio-Rad, Hercules, CA, USA) and performed on a Bio-Rad CFX 96 instrument. Primers [12 (link)–16 (link)] used in this study are shown in Table 1. The reaction procedure was the following: 95 °C, 2 min; 95 °C, 5 s, 40 cycles; 60 °C, 30 s; 95 °C, 5 s; 60 °C, 5 s; 95 °C, 2 min. Three replicates were set for each gene, and 2-^ΔΔCT was used for relative quantitative analysis.Table 1

Primer and probe sequences for qPCR.

Gene		5′3’sequence
β-actin	Forward	GTGACGTTGACATCCGTAAAGA
	Reverse	GCCGGACTCATCGTACTCC
il-1β	Forward	GAA ATG CCA CCT TTT GAC AGT G
	Reverse	TGG ATG CTC TCA TCA GGA CAG
nlrp3	Forward	ATT ACC CGC CCG AGA AAG G
	Reverse	CAT GAG TGT GGC TAG ATC CAA G
cxcl1	Forward	ACT GCA CCC AAA CCG AAG TC
	Reverse	TGG GGA CAC CTT TTA GCA TCT T
cxcl2	Forward	CCA ACC ACC AGG CTA CAG G
	Reverse	GCG TCA CAC TCA AGC TCT G
cxcr2	Forward	ATG CCC TCT ATT CTG CCA GAT
	Reverse	GTG CTC CGG TTG TAT AAG ATG AC

To quantify cytokine levels induced by S. suis in PAM-Tang cells, all the supernatants were removed from the wells and collected on sequential samples of cell cultures, then replaced with fresh medium at 3, 8, 16, and 24 h post-infection (hpi). The levels of porcine IL-18, IL-1β and TNF-α were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits (XIpcc, Shanghai, China; Cusabio, Hangzhou, China) according to the manufacturer’s instructions. The amount of cytokines (ng/L; pg/mL) was calculated according to a standard curve generated from recombinant controls supplied in the kits.
Infected cells at 3, 8, 16, and 24 hpi were collected and total RNA was extracted by RNA Pre-Pure kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Then, total RNA was retro-transcribed into complementary DNA using PrimeScript RT reagent Kit (TaKaRa, Dalian, China). SYBR^® Green (Vazyme Biotech, Nanjing, China) was used to quantify the PCR-amplification products and GADPH was used as a reference for quantitative RT-PCR (qPCR). Primers were used as follows: IL-6-F: 5′-TGGCTACTGCCTTCCCTACC, IL-6-R: 5′-CAGAGATTTTGCCGAGGATG; IL-8-F: 5′-GCTCCCAAGAATTTCTCAGTA, IL-8-R: 5′-CAGCAGCCTAGGGTTGCAAG; GADPH-F: 5′-TCGGAGTGAACGGATTTGGC, GADPH-R 5′-TGCCGTGGGTGGAATCATAC.