Abstract
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.
Highlights
Egg production is one of the most important economic traits and reproduction trait for hens
We found that 18SrRNA ribosomal RNA (18SrRNA), Beta-actin (ACTB), homeoboC8 (HOXC8), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chicken alpha-Aglobingene and chicken alpha-Dglobingene as candidate internal controls may be commonly used in chicken, but these genes changed a lot between various sample panels. 18SrRNA is a member of the ribosomal subunits, which is conservative and easy to use as general primer amplification in all eukaryotic cells
Reverse transcription (RT) of RNA was performed in 30μl reaction volumes using a First Strand Synthesis Kit (QIAGEN, Crawley, UK) based on the manufacturer’s protocol
Summary
Egg production is one of the most important economic traits and reproduction trait for hens. As known the process is complicated and under high coordination in hens, which was jointly regulated by the hypothalamus pituitary gonadal (HPG) axis endocrine hormone and many genes in the ovarian follicles in particular. In addition to the follicles, the mRNA expression profiles of many genes in the other tissues and organs is vital to the influence on the reproductive physiology in chicken, which are involved in regulating the development of the ovarian follicles. The technique of RT-qPCR assay is a sensitive and powerful method to study the differences in gene expression (Liu et al, 2013). It can monitor gene expression by measuring mRNA levels and detect more eRBCA-2019-1256
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