Chromo4 real time pcr system

MC3T3-E1 cells were cultured on TN or TR samples for 7 days. After being illuminated under 5.5 mW/cm² power of UV365 for 30 min, the total RNA of each sample was extracted using Trizol Reagent (Invitrogen, USA). The concentration and purity of total RNA were measured by a microultraviolet spectrophotometer (SMA1000, Merinton, USA). Then, RNA was reverse-transcribed to cDNA using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Japan), and iTaq Universal SYBR Green Supermix (Bio-Rad, USA) was used for PCR reactions. The primer sequences are shown in Table 1. The thermocycling conditions were followed according to the manufacturer's instructions in the Chromo-4 Real-Time PCR System (Bio-Rad, USA). Calculation of the gene copy number was carried out using the −ΔΔCt method [24 (link)].

Total RNA was isolated from tissues by using AxyPrepTM Blood Total RNA MiniPrep Kit (Axygen) according to the manufacturer’s instruction. First-strand complementary DNA (cDNA) was synthesized with RevertAid™ First Stand cDNA Synthesis Kit (Fermentas) using a random hexamar primer. Quantitative PCR was performed through BioRad Chromo4 real-time PCR system. The primer sets for amplifying NEAT1 and reference gen (ACTB) are the following: NEAT1-F: TGG ACT AGCT CAG GGA CTT CAG; NEAT1-R: TCT CCT TGA CCA AGA CTT CCT TC; ACTB-F: GAC CTG ACT GAC TAC CTC ATG AAG AT; ACTB-R: GTC ACA CTT CAT GAT GGA GTT GAA GG. At the end point of PCR cycles, melt curves were made to check product purity. The level of NEAT1 was expressed as a ratio relative to the β-actin messenger RNA (mRNA) in each sample. Exploratory data analysis using scatter plot was applied to visually identify the expression level of target mRNA. Statistical analysis was performed using paired t test or non-parametric test. P values less than 0.05 were considered statistically significant.

Total RNA was extracted from stored tissue with RNAlater using a commercial kit (RNeasy fibrous tissue mini kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions and was reverse transcribed to cDNA. cDNA was synthesized from mRNA using oligo(dT)₂₀ primers and the Super-Script^™ III First-strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). The cDNA samples, primers specific for the target genes (TLR3, IFNβ, IFNγ, Mx1, OAS, IL6, IL8, and IL18), β-actin, and SYBR Premix Ex Taq2 (PR081A, Perfect Real Time; TaKaRa Bio, Inc., Otsu, Japan) were prepared according to the manufacturer’s instructions. The sequences of all the primer pairs used in the quantitative real-time PCR analysis were previously described [29 (link)]. The quantitative real-time PCR analysis was run on a Chromo4 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) with the following cycle parameters: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. The differences in gene expression were calculated by the 2^–ΔΔCt method and were expressed as the fold change in gene expression as previously described [30 (link)]. β-Actin was used as the endogenous control for normalization of target gene expression [29 (link)]. The mean expression ± standard deviation was expressed as fold change and was compared to the expression in uninfected chickens.

To validate RNA‐seq data, expression of selected DEGs (including defence marker genes CRK, WRKY, and GST) was analyzed by qRT‐PCR. To perform qRT‐PCR, first‐strand cDNA was synthesized from 2 mg of DNase‐treated total RNA using oligo‐dT primers and Moloney murine leukaemia virus reverse transcriptase (MMLV‐RT, Enzynomics, Daejeon, South Korea). PCR reactions were performed according to the manufacturer’s instructions. Expression of DEGs was analyzed using the primers listed in Table S1. A Chromo4 Real‐Time PCR system (Bio‐Rad, CA, USA) was used for qRT‐PCR. Reaction mixtures contained cDNA template, iQTM SYBR^® Green Supermix (Bio‐Rad, CA, USA) and 10 pM of each primer. Thermocycler parameters used for qRT‐PCR were as follows: initial polymerase activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 60 sec, and extension at 72°C for 30 sec. Conditions were determined by comparing threshold values in a series of dilutions of the reverse‐transcribed product, a non‐reverse‐transcribed template control, and a non‐template control for each primer pair. Relative RNA levels were calibrated and normalized relative to the level of AtActin2 mRNA.

Total RNA was extracted from tomato leaves harvested at 0 and 12 h after pathogen inoculation, and first-strand cDNA was synthesized as described previously [55 (link)]. Then, quantitative real-time PCR (qRT-PCR) was performed on the Chromo4 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using the cDNA template, iQTM SYBR® Green Supermix (Bio-Rad), and 10-pM sequence-specific primers (Table S2) under the following conditions: initial polymerase activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The expression level of each gene was calibrated and normalized relative to that of Ubiquitin3 mRNA. All qRT-PCR experiments were performed in triplicate.