Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus ).

Carlos S Nascimento,Renata S Mann,Roberta P M Fernandes,Ana Paula G Pinto,Claudson Brito,Mike V Dodson,Marcio S Duarte,Simone E F Guimarães,Leandro T Barbosa,Haniel C Oliveira,Ramona Natacha Pena I Subirà

doi:10.1371/journal.pone.0127935

Abstract

Thirteen reference genes were investigated to determine their stability to be used as a housekeeping in gene expression studies in skeletal muscle of chickens. Five different algorithms were used for ranking of reference genes and results suggested that individual rankings of the genes differed among them. The stability of the expression of reference genes were validated using samples obtained from the Pectoralis major muscle in chicken. Samples were obtained from chickens in different development periods post hatch and under different nutritional diets. For gene expression calculation the ΔΔCt approach was applied to compare relative expression of pairs of genes within each of 52 samples when normalized to mitochondrially encoded cytochrome c oxidase II (MT-CO2) target gene. Our findings showed that hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are the most stable reference genes while transferrin receptor (TFRC) and beta-2-microglobulin (B2M) ranked as the least stable genes in the Pectoralis major muscle of chickens. Moreover, our results revealed that HMBS and HPRT1 gene expression did not change due to dietary variations and thus it is recommended for accurate normalization of RT-qPCR data in chicken Pectoralis major muscle.

Highlights

Quantitative real-time PCR (RT-qPCR) is a gold-standard biotechnique for gene expression analyses but requires a robust reference genes to correct for technical variation within th
Samples were obtained from chickens in different development periods post hatch and under different nutritional diets Our results revealed that hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) gene are most stable and recommended for accurate normalization of RT-qPCR data while transferrin receptor (TFRC) and B2M varied across the experimental conditions and are not recommended to normalize gene expression data
The no-template controls (NTC) that was included in all RT-qPCR assays did not show any positive quantification cycle (Cq) value throughout the study

Summary

Methods

Ethical approvalAll experiments were approved by the Universidade Federal de Sergipe Animal Ethics Committee and Brazilian national standards for animal research.Broiler, experimental condition and muscle samplingA total of one hundred sixty-eight 1-day-old Cobb male broilers were obtained from a commercial hatchery after being vaccinated against Marek’s disease and Newcastle disease. Broilers were allocated in floor pens until day 7. Experimental diets were based on corn and soybean meal and were formulated using the food chemical composition values and nutritional requirements for the average performance of male broilers [14]. Experimental diets were maize-soyabean meal-based supplemented with four levels of L-Lys (12.71, 11.82, 10.99 or 10.22 g/kg) during the growing phase, and four levels of L-Lys (11.25, 10.46, 9.72 or 9.030 g/kg g/kg) during the finishing phase. A total of 52 broilers (4 male in each group) were randomly sampled at 8, 21, 35 and 42 days post hatch and slaughtered. The control group was composed by broilers supplemented with 0 g/kg of L-Lys and slaughtered at 8 d post hatch. Samples were placed at 4°C for 12 h and stored at 70°C prior to total RNA isolation

Results

Discussion

Conclusion