Sybr green

B16.F10 melanoma cells were cultured in the presence or absence of IL-33 (100 ng/mL) for 4 h. Total RNA was extracted from tumor cells by using TRIsure reagent (Bioline, London, UK). mRNA was reverse transcribed by means of Tetro cDNA Synthesis Kit (Bioline). Quantitative reverse transcription-PCR (qPCR) with forward and reverse primers for CCL5, CCL24 and HPRT (Eurofins Genomics, Ebersberg, Germany) [6 (link)] was performed using Sensimix Plus SYBR Kit containing the fluorescent dye SYBR Green (Bioline) and by means of an ABI 7500 Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, Ma, USA). Triplicates were performed for each experimental point. Data were normalized to HPRT (2-ΔCt method) and presented as fold change expression vs. control.

Total RNA was extracted from cells after the specific treatment, at indicated time points, using RNeasy Mini Kit (Qiagen^TM, Hilden, Germany) as per the manufacturer’s protocol (n = 9). Briefly, 1 μg of RNA was reverse transcribed using the ReverTraAce^® qPCR RT Master Mix with gDNA remover (Toyobo^TM, Osaka, Japan) as per the manufacturer’s protocol. The quality of the cDNA synthesized was evaluated via Biodrop 260/280 nm (BioRad, Hercules, CA, USA). The resulting cDNA was diluted in a 1:10 ratio, and 150 ng of the cDNA was used as a template for SYBR green (Bioline, London, UK) based real-time PCR amplification reaction. The amplification was performed using the following conditions: one cycle of 95 °C for 3 min, 35–40 cycles of 95 °C for 30 s, and respective annealing for 45 s followed by melt curve analysis. Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as internal control, and the relative target gene fold change in NP and hnRNPA1 mRNAs was calculated using the delta-delta threshold cycle 2^−ΔΔCt method (in reference to the control) [37 (link)]. Statistical analysis was performed to compare the difference between two different treatment groups using the student’s t-test (two-tailed), respectively.
The primers used for real-time based analysis are tabulated in Table 1.

Transcript levels of defense-related genes in response to B. cinerea or Pst were analyzed by qPCR. Leaves were ground in liquid N₂, and total RNA was extracted with the Spectrum^TM Plant Total RNA Kit (Sigma-Aldrich, Saint Louis, MO, United States). The isolated RNA was treated with deoxyribonuclease I enzyme (Sigma-Aldrich) to remove remaining DNA. Two micrograms of purified RNA were used for reverse transcription reactions with the Omniscript Reverse Transcription Kit (Qiagen). The qPCR mixture contained 7.5 μL of SYBR Green (Bioline), 5 μL of cDNA (corresponding to 100 ng RNA), and 0.5 μL of 10 μM forward and reverse primers (Supplementary Table 1). The final volume was completed with DEPC-treated Water (0.1% diethylpyrocarbonate) to 15 μL. The qPCR was done as follows: 10 min at 95°C initial denaturation and 40 cycles (95°C for 15 s, 60°C for 1 min and 72°C for 30 s). Runs were performed on a MIC qPCR machine (Bio Molecular Systems). Transcript levels were normalized against the expG gene (AT4G26410). The analysis was accomplished based on cycle threshold method (2^(–ΔΔCt); Rao et al., 2013 (link)). Three biological replicates were performed for each sample.

RNA was extracted from single adult organs using TRIzol (Life Technologies). A qScript cDNA Synthesis Kit (Quantabio) was used to generate cDNA. Quantitative real-time PCR (qPCR)-based quantification of galt, galk1 (galactokinase), gale (UDP-galactose 4′-epimerase), and akr1b1 (aldose reductase) expression was conducted, with ef1a as a reference gene (Tang et al 2007 (link)). Primer sequences are available on request. SYBR® green (Bioline) was used for all qPCR experiments. Samples, obtained from two independent RNA extractions were measured five times in total.

Quantitative real-time PCR was used to assess the expression of proinflammatory cytokine genes, including IL1β, IL6, CXCL8, and TNF, IFNB1, and the pattern recognition receptors TLR3, TLR4, and TLR7 (toll-like receptors for viruses and bacteria) as well as DDX58 and IFIH1 (RIG-I-like receptors (RLRs) for viruses). Amplified products were detected using SYBR green (BioLine, Taunton, MA, USA). Primers were designed in-house (Table 1). All primer pairs have passed the BLAST specificity screen (specifically bind to corresponding target gene). Reactions were performed using a LightCycler 480 (Roche Diagnostics, Indianapolis, IN, USA), with gene expression normalized to the housekeeping gene hypoxanthine-guanine phosphoribosyl-transferase (HPRT). All samples were assessed in triplicate.
Additional details of the methods for RNA isolation, cDNA synthesis, and real-time PCR are presented in the Supplementary Material file; .

RT-qPCR assays were performed in conformation with MIQE guidelines (http://www.rdml.org/miqe.php). One μg of total RNA was reversely transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's recommendations using Random Primers. qPCR assays were performed using a Biorad CFX384/qPCR Instrument, from 5 ng of the corresponding cDNAs in 10 μl final volume with SYBR Green (Bioline) reagents: 95°C 2 min, then 40 cycles at 95°C 5 s/60°C 10 s/72°C 15 s, followed by a dissociation step. Primers for qPCR analysis used in this work were manufactured by Eurofinsgenomics with the corresponding sequences listed in Supplementary Table S4. PCR efficiency was calculated from amplification slope for each couple of primers; control RT minus assays were carried out in order to verify specific amplification of cDNAs with respect to genomic DNA. Three technical replicates were performed for each qPCR assay. Relative quantification of mRNA expression was calculated using the ΔC_T method with respect to three reference genes (Ppib, Hmbs, Rplp0).