Evaluation and Validation of the Six Housekeeping Genes for Normalizing Mrna Expression in the Ovarian Follicles and Several Tissues in Chicken

Abstract

Expression of housekeeping genes is relatively constant in different tissues and cells by RT-qPCR analysis. Housekeeping genes (HGs) are usually utilized as the reference to evaluate and compare mRNA expression abundances of target genes in different cells or tissues sampled. However, the expression stabilities of different HGs in diverse samples may appear divergence. Currently, there is no exact reference data of HGs in hen ovarian follicular tissues during egg-laying period available yet. In this study, we detected the expression of 18SrRNA, ACTB, HOXC8, GAPDH, alpha-A, and alpha-D mRNA in the varied-size ovarian follicles (1-8 mm in diameter and F5), hearts, livers, spleens, lungs, and breast muscles of the laying hens by RT-qPCR, to analyze the results via Ct value, geNorm, Normfinder, and Bestkeeper. The data showed that the expression levels of 18SrRNA, alpha-A, and alpha-D transcripts were more significantly stable than the other three genes for normalizing mRNA expression in the hen ovarian follicles examination. Moreover, alpha-D, 18SrRNA, and alpha-A were also most suitable for the expression normalization in the tissues of the heart, liver, spleen, lung and breast muscle. In contrast, 18SrRNA has the most stable mRNA expression levels in all tissues sampled, so it can serve as an excellent inner control for the evaluation of the transcription levels in chickens. It is a remarkable fact that HOXC8 as a candidate reference should be avoided. Our study establishes a set of stably expressed candidate inner references in the hen ovarian follicles and several tissues, it firstly provided an exact data for validation of the inner references in normalizing transcription levels of a target gene in chickens.

Antioxidant Status of Broiler Chickens Fed Diets Supplemented with Vinification By-Products: A Valorization Approach

“…The relative mRNA expression levels for the target genes were quantified with a StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as described by Mavrommatis et al [ 35 ]. GAPDH and ACTB were used as housekeeping genes to normalize the cDNA template concentrations [ 36 ]. The relative expression levels of the target genes were performed as described by Mavrommatis et al [ 35 ], while the primers’ efficiency was calculated by employing the linear regression method on the log (fluorescence) per cycle number (ΔRn) using the LinRegPCR software [ 37 ].…”

Immune-Related Gene Expression Profiling of Broiler Chickens Fed Diets Supplemented with Vinification Byproducts: A Valorization Approach II

“…PCR program initiated at 95 °C for 8 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for one min. The geometrical mean of GAPDH and ACTB were used to normalize the cDNA template concentrations as has been previously indicated [ 20 ]. The relative expression levels of the target genes were performed as described by Mavrommatis et al [ 21 ], while PCR efficiency was determined by employing the linear regression method on the log (fluorescence) per cycle number (ΔRn) using the LinRegPCR software applying the default settings [ 22 ].…”

The Impact of Hydrated Aluminosilicates Supplemented in Litter and Feed on Chicken Growth, Muscle Traits and Gene Expression in the Intestinal Mucosa

“…Primer sequences were derived from the literature data, our previous published scientific reports or designed based on a cDNA nucleotide sequence using NCBI Primer Blast [ 22 ]. The selection of the reference genes was based on the literature [ 23 , 24 ] and our previous molecular analyzes [ 21 ]. Analysis included the geometric mean of two independent reference genes ( ACTB and G6PDH ).…”