Cfx96 real time system

Total RNA was extracted from the cultured PECs cells by using TRIzol reagent (Invitrogen, Carlsbad, CA.). Reverse transcription was performed with Oligo 18T primers and reverse transcription reagents according to the manufacturer's protocol (TaKaRa, Shiga, Japan). Quantitative real-time polymerase chain reaction (PCR) was performed with mRNA special primers. The following primers were used for the PCR assay: for NOX4, 5′-TATCCAGTCCTTCCGTTGGTT-3′ (forward) and 5′-CTGAGGTACAGCTGGATGTTGA-3′ (reverse); for SOD2, 5′-GAGAAGTACCAGGAGGCGTTG-3′ (forward) and 5′-GAGCCTTGGACACCAACAGAT-3′ (reverse) and for NRF-2, 5′-AAACCAGTGGATCTGCCAAC-3′(forward) and 5′-GACCGGGAATATCAGGAACA-3′(reverse). PCR reactions were performed on a BIO-RAD CFX-96 Real Time system with SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 57°C for 30 seconds, and 75°C for 10 seconds, after which melt curve analysis was performed at once from 65°C to 95°C. All reactions were performed in triplicate and the average cycle threshold (Ct) values greater than 38 were treated as negative. The level of GAPDH mRNA was used as an internal control.

Total RNA was extracted using the Trizol method, according to the manufacturer’s instructions (Invitrogen, USA). After reverse transcription was performed, quantitative real-time PCR (qRT-PCR) was analyzed in a BIO-RAD CFX96 Real-Time System, using SYBR^® Premix Ex Taq™ II (Takara, Japan). The primers were designed using NCBI primer-blast and synthesized by Sangon biotechnology (Shanghai, China) as follows: PGC-1α (forward: GGA CAC GAG GAA AGG AAG ACT A; reverse: GTA GCA CTG GCT TGA ATC TGT G) and β-actin (forward: CCC ATC TAT GAG GGT TAC GC; reverse: TTT AAT GTC ACG CAC GAT TTC). The specificity of PCR products was guaranteed by melting curve analysis. The expression of PGC-1α was normalized to β-actin, and the gene expression was comparatively analyzed using the 2^−ΔΔCt method.

The 24 h post-sham/MI-operated tissues of hearts were harvested and ground into powder in liquid nitrogen. The powdered tissues were lysed and chromatin was extracted using the SimpleChIP^® Plus Sonication Chromatin IP Kit (Cell Signaling Technology, Cat No. #56383). The chromatin was sheared by sonication and genomic DNA was immunoprecipitated with a ChIP-grade anti-H3K4me1 antibody (Abcam, Cat No. ab8895) following the SimpleChIP^® Plus Sonication Chromatin IP Kit. The qPCR was performed by CFX96™ Real-Time System, according to the instructions of TB Green^® Premix (Takara, Cat No. RR820A). The quantification was calculated using the 2^−ΔΔCt method, and input DNA was used as a normalized reference. The primers were shown in Supplementary Table S3.

Total RNA from cells was extracted using the RNAiso Plus (Takara Bio Inc., Otsu, Japan). The cDNAs were synthesized by PrimeScript^™ RT reagent Kit with gDNA Eraser (Takara). Expression of the genes of interest was examined through a Bio-Rad CFX96^™ Real-Time System with SYBR^® Premix Ex Taq^™ II (Takara). GAPDH was used as an internal control in quantitative analysis. Primers used in real-time PCR analyses are listed in S1 Table. PCR amplification protocol involved 40 cycles of denaturation at 95°C for 10 seconds, primer annealing at 59°C for 20 seconds and primer extension at 72°C for 10 seconds. Each sample was analyzed at least in triplicate. The threshold cycle number (Ct) values of genes were determined. Gene expression level was normalized to GAPDH and presented as the fold change (2^−ΔΔCt) above control group.

Mouse lungs were removed at different time points after pneumococcal 19F challenge, and total RNA was extracted using RNAiso Plus reagent (Takara Bio, Dalian, China) following the manufacturer’s instructions. For reverse transcription, 1 µg of total RNA was reverse-transcribed into cDNA, and the PrimeScript™ RT Reagent Kit (Takara Bio) was used for reverse transcription. PCR amplification was performed on the Bio-Rad CFX96 Real-Time system using SYBR Premix Ex Taq™ (Takara Bio). The 2^−ΔΔCt method was used to determine the specific Ct value of each target gene. Quantitative real-time PCR for each target gene were repeated three times. The PCR primers were synthesized by Sangon (Shanghai, China) and the sequences are listed in Table S1 in Supplementary Material. All genes were murine in origin.

Total RNA from whole honeybees was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The first strand of cDNA was acquired using a HiScript^® II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China) following the supplier’s instructions. The qRT-PCR analyses were carried out using SYBR^®Premix Ex Taq^TM (Tli RNaseH Plus) (TaKaRa, Dalian, China) and a CFX96^TM Real-Time System. The efficiency values of all pairs of primers used for qRT-PCR were all within 90–110%. Each pair of qRT-PCR primers had a single peak, and their correlation coefficients (R²) were approaching 1. The specific sequences of qRT-PCR primers are presented in Supplementary Table S2. The β-actin gene (GenBank accession no. HM640276.1) was used as a standard to normalize other gene expression levels as it is stably expressed in honeybees (Lourenço et al., 2008 (link); Yao et al., 2014 (link)). For each treatment, at least three biological replicates were performed. The 2^-ΔΔCT method and CFX Manager software (version 3.1) were used to analyze the relative levels of gene expression.