Rt pcr system

Real time PCR (RT-PCR) assay was conducted to assess expression of alpha hemolysin (hla) under SNPs treatment. RNA isolation and cDNA synthesis were conducted following general procedure. Primer pair sequences for real time PCR of the virulence factor are 5′-TGAATCCTGTCGCTAATG-3′ as the forward primer; and 5′-TATCATCCGACCTTTCACT-3′ as the reverse primer (15). 16s RNA was used as housekeeping (reference) gene and internal control in RT-PCR assay. Forward and reverse sequences of the reference gene were 5′-CGTGCTACAATGGACAATACAAA-3′ and 5′-ATCTACGATTACTAGCGATTCCA-3′, respectively ^{15 (link)}. Expression of the virulence gene (hla) was quantitatively analyzed using a RT-PCR system (BioRad). RT-PCR was carried out in a 20 μl reaction volume containing 0.5 μM of each primer and 10 μl of SYBR green real time PCR master mix (Genet Bio, South Korea). Quantitative RT-PCR experiments were performed in duplicate for each sample. The RT-PCR data were analyzed by the ΔΔCt method as described by Xiang et al^{15 (link)}.

Cutaneous total RNA extracted with TRIzol reagent (Vazyme, Nanjing, China) was used to generate complementary DNA obtained by reverse transcription with commercial kits (Takara, Tokyo, Japan). Quantitative PCR was carried out using an RT-PCR System (Bio-Rad, Hercules, CA, USA). The gene expression was quantified with the method of 2^−ΔΔCt. The primers of the determined genes are shown in Table 2.

The expression of nifH, CAT, PAL, SOD, CHI and GLU genes in leaf tissues of sugarcane during plant-microorganism interaction was analyzed under a greenhouse condition after inoculation of selected strains (CY5 and CA1) in GT11 and GXB9 sugarcane varieties. Both sugarcane varieties were provided by Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China. The relative expression of all genes was measured by calculating the difference in the expression level of the inoculated sample and the control at 60 and 120 days with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene.
Real-time quantitative PCR (RT-qPCR) was conducted with SYBR Premix Ex Tap™II (TaKaRa, Japan) and the reaction was completed in RT-PCR System (Bio-Rad, USA). The total reaction volume was 20 μL, the composition of the reaction mixtures and conditions were followed by Niu et al. [98 ]. The list of primers used is presented in Table 5. The specificity of the reaction was confirmed by melting curve analysis at the end of the amplification and 2^−△△Ct method was used for quantification of relative gene expression [107 (link)]. Each RT-qPCR experiment was conducted in triplicate.

To quantify the cDNA of Pst, the standard curves were first created with the Pst translation elongation factor gene via real-time PCR analysis. The threshold cycles (Cq) were plotted against the concentration of cDNA of the Pst race CYR31 (7.215, 3.608, 2.405, 1.804, 0.902 and 0.722 ng/μl) (Fig. S5). Dissociation curves were generated for each reaction to ensure specific amplification. The quantification of cDNA was performed using a RT-PCR System (Bio-Rad, Hercules, CA, USA). Three independent biological replicates were performed for each experiment.

The levels of miR-132-3p in MSCs, MSC-EXs, ECs, and microvessels were measured by quantitative real-time PCR. Total miRs were extracted by using the miRNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. The miR-132-3p cDNA was synthesized using the Hairpin-itTM miR RT-PCR Quantitation kit (GenePharma, Shanghai, China) under 25 °C for 30 min, 42 °C for 30 min, and 85 °C for 5 min. Real-time PCR was conducted on a RT-PCR system (Bio-Rad). The parameters were 95 °C for 3 min and 40 cycles performed at 95 °C for 12 s and 60 °C for 40 s. PCR primers were as follows: 5-CCAGCATAACAGTCTACAGCCA-3 and 5-TATGGTTGTTCACGACTCCTTCAC-3 for miR-132-3p, and 5-CTCGCT TCGGCAGCACA-3 and 5-AACGCT TCACGAATTTGCGT-3 for U6. The level of miR-132-3p was normalized to U6. The relative quantification of the gene expression was determined using the comparative CT method (2^−ΔΔCt).

Total RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions, and was quantified by a NanoVue Plus spectrophotometer (GE Healthcare, Chicago, IN, USA). MiRNAs were reverse transcribed using a miRNA First Strand cDNA Synthesis Kit (Tailing Reaction, Sangon Biotech, Shanghai, China), while mRNAs were reverse transcribed into cDNA using Reverse Transcription Reagents (TaKaRa Bio, Shiga, Japan), according to the manufacturer’s protocols. Quantitative real-time (RT)-PCR was conducted using TB Green^TM Premix Ex Taq^TM (TaKaRa Bio) on a RT-PCR system (Bio-Rad Laboratories). U6 and GAPDH were used as internal controls to determine the relative expression of miRNAs and mRNAs, respectively. The relative expression was reported as a 2^−△△Ct value. To construct a miRNA panel, 92 potential miRNAs most likely to be relevant to inflammation were selected based on the miRNA list provided by Qiagen (Valencia, CA, USA). The primer sequences for all miRNAs and mRNAs are shown in Supplementary Table 1.

20 mg RNA of mice skin samples (physical homogenization) and cells were extracted by TRIzol (Invitrogen, USA). After chloroform extraction, isopropyl alcohol was added and allowed to stand before centrifugation. Next, the RNA was washed with ethanol, centrifuged and dried, and 25 μL DEPC water was added. The purity and concentration of RNA were measured by NanoDrop (ThermoFisher, USA). Genomic DNA removal was performed and complementary DNA was reversed transcribed (Accurate Biology, China). Quantitative realtime polymerase chain reaction (PCR) was performed by SYBR Green method (TransGen Biotech, China) using RT-PCR System (Bio-Rad, CA). Primers are described in the (Additional file 2: Table S3). The mRNA expression levels were figured by 2^−ΔΔCT method. Data were normalized to β-actin or GAPDH and compared with controls.