Beacon designer

mRNA was extracted from snap‐frozen 16h‐stimulated moDCs using a NucleoSpin® RNA Plus kit (Macherey‐Nagel, Germany) in combination with the rDNAse set (Macherey‐Nagel, Germany) to remove contaminating DNA. cDNA was synthesized using an iScript™ advanced kit (Bio‐Rad, USA) according to the manufacturer's protocol in the PTC‐100 Programmable Thermal Controller (MJ research, PTC‐100). cDNA and mRNA samples were stored at ‐80°C. Quantitative analysis was conducted on a CFX96 Real‐Time C1000 Thermal Cycler detection system with the use of an IQ™ SYBR® Green Supermix, according to manufacturer's protocol (both from Bio‐Rad). Primers for the genes of interest are listed in Table 1. Primers (produced by Biolegend, USA) were designed with Primerblast (NCBI, USA) and Beacon Designer (Premier Biosoft International, USA). Primers were validated based on the efficiency and R². Primer specificity was tested by running the products on a 2% agarose gel (70 minutes; 110V). The expression of the genes of interest was determined using 12.5ng cDNA and SYBR green (Bio‐rad, USA). The RT‐PCR protocol consists of 3 min at 95°C followed by 10 s at 95°C and 30 s at the optimal primer annealing temperature as determined beforehand. The samples were measured in duplo and mean Ct values were used to determine the relative expression using the Pflaff method and β‐actin as a reference gene.

Total of 19 unigenes were randomly chosen for validating the expression of DEGs presented in the transcriptomes and detecting the variation of their expressions in samples using qPCR. Primers for unigenes were designed by Primer3 version 0.4.0^{55 (link)} and Beacon designer version 8.14 (PREMIER Biosoft, Palo Alto, CA, USA), suitable primers were screened out for the final qPCR experiment by estimating its specificity, amplification efficiency, etc. The cDNA of each sample prepared was diluted 1:10 with nuclease-free water before qPCR analysis. Each reaction was performed in total of 15 μl mixture containing 7.5 μl of 2 × SYBR® Green ROX qPCR mastermix (QIAGEN, USA), 1 μl of cDNA template, 0.5 μM of each primer, with nuclease-free water supplied to final volume. The qPCR was carried out in 96-well blocks with ABI PRISM® 7000 Sequence Detection System (Applied Biosystems). qPCR amplification was performed under a program: 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Dissociation curve was obtained by heating the amplicon from 60 to 95 °C. Technical and biological triplicates were applied for qPCR reaction. Mean relative expression ratio was calculated using the 2^−ΔC(t) method^{56 (link)} with one stably expressed gene as reference gene.

Total RNA was extracted from the ear using TRIzol reagents (Gibco-Life Technologies, ThermoFisher Scientific Inc.). The reverse transcription reaction contained 1 μg of RNA and was performed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The blastx (National Center for Biotechnology Information, Bethesda, MD, USA), Primer3Plus (Whitehead Institute for Biomedical Research, Cambridge, MA, USA), and Beacon Designer (Premier Biosoft, Palo Alto, CA, USA) programs were used for the design of primers specific for the desired sequences. They were analyzed for optimal annealing and melting temperatures, the optimal amount of cytosine and guanine bases, and minimal formation of secondary structures.

A Biorad CFX384 (Biorad, Czech Republic) was used for all qPCR measurements. To each reaction (10 µl) containing 5 µl iQ SYBR Green Supermix (BioRad) and 300 nM of each primer (EastPort, Czech Republic) was added 2 µl of 20-fold diluted preamplified cDNA. The temperature profile was 95°C for 3 min followed by 40 cycles of amplification (95°C for 15 s, 60°C for 20 s and 72°C for 20 s). All samples were analyzed by melting curve analysis. Primers were designed using BeaconDesigner (version 7.91, Premier Biosoft International) as described previously [15] (link). The primer sequences are shown in Table 1.