Lightcycler 96 instrument qrt pcr system

To verify the reliability and accuracy of the sequencing results, five DEGs (FABP3, CYP7A1, ANKRD22, SCD1, and PCK1) that were significantly enriched in the PPAR pathway were selected for qRT-PCR verification. Seven liver samples were selected from the HA and control groups. Liver tissue was used to isolate the RNA using TRIzol Reagent (Takara Bio Inc. Otsu, Shiga, Japan). The total RNA purity and concentration were measured using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA integrity was checked by 1.5% agarose gel electrophoresis. The A260/A280 ratio was expected to range from 1.8 to 2.0. cDNA synthesis was performed using a two-step method offered by the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara Bio Inc. Otsu, Shiga, Japan). All specific primers for qRT-PCR (Supplementary Table S1) were designed using Primer 5.0 and synthesized by Sangon Biotech (Shanghai, China). The expression levels of the selected DEGs were measured using the SYBR Primer Ex Taq™ II Kit (Takara Bio Inc. Otsu, Shiga, Japan) in a LightCycler 96 instrument qRT-PCR system (Roche Molecular Biochemicals, Mannheim, BW, Germany). The results were normalized to the expression levels of chicken β-actin using the 2^−ΔΔCt method for quantification. All experiments were performed in triplicate.

For the qRT-PCR analysis of genes, reverse transcription was performed using a PrimerScript^TM RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. qRT-PCR with iTaq^TM Universal SYBR^® Green Supermix Kit (Bio-Rad Laboratories Inc., Waltham, MA, United States) was performed with a LightCycler^® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) as follows: 95°C for 3 min; 40 cycles of 95°C for 10 s, annealing at 60°C for 30 s, and 72°C for 30 s, and 72°C for 1 min. The data were analyzed with the 2^-ΔΔCt method. The chicken GAPDH gene was used as a reference gene for normalization of target gene data. The sequences of qRT-PCR primers are listed in Supplementary Table S1.
For the qRT-PCR analysis of miRNAs, reverse transcription was performed as for mRNA, except that the miRNA was reverse transcribed with Bulge-loop miRNA qRT-PCR Primer Sets (RiboBio, Guangzhou, China). The qRT-PCR process and the data analysis methods were the same as for mRNA. The primers specific to miRNAs were designed by RiboBio. Chicken U6 small nuclear RNA was used as an internal control for normalization of miRNA data.

To corroborate the RNA-Seq results, we randomly selected 10 genes from the SDEGs for qRT-PCR validation, including five upregulated genes and five down-regulated genes. Chicken spleen tissue total RNA was extracted, and a reverse transcription reaction was carried out using a primerScriptTM RT kit (Takara, Kyoto, Japan) to obtain cDNA for qRT-PCR. The reaction was carried out using the LightCycler^® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) and SYBR^® PremixEx TaqTM kit (Takara, Kyoto, Japan). The qRT-PCR amplification procedure was as follows—95 °C for 3 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and extended at 72 °C for 10 min. In addition, before the gene quantification, three common chicken housekeeping genes (B2M, β-actin and GAPDH) were tested [16 (link)] and evaluated using GeNorm [17 (link)]. Since GAPDH showed a lower M value and was stable in Gushi chickens [18 (link)], it was used as an internal reference gene. The relative expression changes of the genes were calculated using the 2^−ΔΔCt method. The qRT-PCR samples were the same as the RNA-seq samples, with three biological repeats for each group and three repetitions for each sample. The primer sequences are listed in (Additional File 3: Table S2).