Dreamtaq green pcr master mix

4 × 10⁶ Brn4-overexpressing N2a cells were needed for each immunoprecipitation. The ChIP assay was carried out according to the manufacturer's instructions (Cell Signaling Technology). Briefly, the cells were cross-linked by 1% formaldehyde, and then the chromatin was digested to 150-900 bp DNA-protein fragments by micrococcal nuclease and ultrasonic treatment. After respective incubation with different immunoprecipitating antibodies overnight at 4°C, elution from antibody/protein G agarose beads, and reversal of cross-links, the DNA was purified using spin columns and quantified by PCR. PCR was performed with DreamTaq Green PCR Master Mix (Thermo Fisher Scientific) according to the manufacturer's protocol using the following PCR conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 40 s. The results were visualized using 1% agarose gel electrophoresis. The sequences of the primers used in the study are in Table 1.

cDNA samples used this study were previously published²² (link) and PCR was performed with DreamTaq Green PCR Master Mix (Thermo Scientific, Gaithersburg, MD, USA) under the following conditions: denaturation at 98°C for 3 min; 40 cycles of 98°C for 30 s, 58°C for 30s, and 72°C for 90 s, and final extension at 72°C for 10 min. The PCR products were visualized by electrophoresis through a 2% agarose gel. The PCR primers for TLR4, LST1, and CLEC7A of splicing variants are shown in Figure S6A.

Nucleotide sequences corresponding to P. ovale LDH genes were amplified with the primers LDHovD21 (5′-GTTCTCGTTGGTCAGGAATGATA-3′) and LDHovC915 (5′-GGCATCATCAAACATCTTCTTTTCT-3′) by conventional PCR using Dream Taq Green PCR Master Mix (Thermo Scientific). Primer design and PCR conditions were based on a previous publication [20 (link)]. The PCR products were sequenced by Genescript Biological Technology Co., Ltd. (Nanjing, China). Nucleotide sequences of P. ovale LDHs were aligned using BioEdit software and compared with available P. ovale sequences in GenBank (accession number AY486058) and a paper by Bauffe et. al.[20 (link)]. Moreover, amino acid sequences were derived using GeneDoc software and also compared with available P. ovale sequences.

Total RNA was extracted with TRIzol reagent (Gibco, Grand Island, NY, USA) either in niacin, LPS or in combination. Total RNA (1 μg) was subjected to reverse transcription into cDNA synthesis using reverse transcriptase enzyme in a total 20 μL volume with the instructions described in kit manuals (BIO-RAD, Carlsbad, CA, USA). β-actin mRNA was used as the housekeeping gene for internal control. For PCR, briefly, each reaction volume was 25 μL containing 2 μL cDNA template, 10 pmol of each primer (forward and reverse), appropriate amount of DNAse/RNAse free water and 12.5 μL of Thermo Scientific Dream Taq Green PCR master mix (Waltham, MA, USA). The PCR cycling condition was 30 s of denaturation at 95 °C, 30 s of annealing at 55 °C, and 30 s of elongation at 72 °C over 30 cycles for all reactions. The following Primers were used in this study:
TNF-α Forward: 5′- ATA GCT CCC AGA AAA GCA AGC-3′; TNF-α Reverse: 5′- CAC CCC GAA GTT CAG TAG ACA-3′; IL-6 Forward: 5′- TGG AGT CAC AGA AGG AGT GGC TAA G-3′; IL-6 Reverse: 5′- TCT GAC CAC AGT GAG GAA TGT CCA C-3′; IL-1β Forward: 5′-GCC TTG GGC CTC AAA GGA AAG AAT C-3′; IL-1β Reverse: 5′-GGA AGA CAC AGA TTC CAT GGT GAA G-3′; β-actin Forward: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′; β-actin Reverse: 5′-GGA TGC CAC AGG ATT CCA TAC CCA-3′.

3 SD rats in each group were used in RT-PCR for each experiment. Total extracted RNA was quantified and quality checked using Nanodrop 2000 (Thermo Scientific, USA). LncRNAs were reverse transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) at 65 °C for 5 min, 42 °C for 60 min, and 72 °C for 5 min. The sequence of primers for PCR amplification of Gm21284 as follows: sense 5′-AAGAGACTGTGAGCACCAGGAG-3′ and antisense 5′-TCTCAGCAGAGTCAAGCCATTC-3′, designed and synthesized by RiboBio (Guangdong, China). Quantitative real-time PCR and semi-quantitative PCR were conducted using SYBR Green Master Mix (Roche, Germany) and Dream Taq Green PCR Master Mix (Thermo Scientific), respectively. PCR reactions were performed at 95 °C for 40 s, 59 °C for 40 s, repeated within 40 cycles. GAPDH and U6 were used as endogenous controls. Fold changes were calculated using the relative quantification 2^−∆∆Ct method. All experiments were performed in triplicate.

Total RNA from HDFs were isolated using the SV Total RNA Isolation System following the manufacturer’s instructions (Promega, Madison, WI, USA), and cDNA synthesis was performed using the SUPERSCRIPT III First-Strand System kit (Thermo Fisher Scientific, Waltham, MA, USA). Hot start PCR was performed using 2 ng cDNA in a total volume of 50 μL containing DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA).
The primer sequences were as follows: human-Panx1, Forward: (5′-GCTATTACTTCAGCCTCTCC-3′), Reverse: (5′-CAGTATCTCCACCAAGAACC-3′); human-Panx3, Forward: (5′-AGGGCTGCTAAGTGATGAGA-3′), Reverse: (5′-GAGGTGTTTGGGTTTTGAGG-3′). PCR reactions were performed using 40 cycles and amplified PCR products were visualized in a 3% agarose gel.