Investigation of chicken housekeeping genes using next-generation sequencing data

Abstract

Accurate normalization of the gene expression assays, using housekeeping genes (HKGs), is critically necessary. To do so, selection of a proper set of HKGs for a specific experiment is of great importance. Despite many studies, there is no consensus about the suitable set of HKGs for implementing in the quantitative real-time PCR analyses of chicken tissues. A limited number of HKGs have been widely used. However, wide utilization of a little number of HKGs for all tissues is challenging. The emergence of high-throughput gene expression RNA-seq data has enabled the simultaneous comparison of the stability of multiple HKGs. Therefore, employing the average coefficient of variations of at least three datasets per tissue, we sorted all reliably expressed genes (REGs; with FPKM ≥ 1 in at least one sample) and introduced the top 10 most suitable and stable reference genes for each of the 16 chicken tissues. We evaluated the consistency of the results of five tissues using the same methodology on other datasets. Furthermore, we assessed 96 previously widely used HKGs (WU-HKGs) in order to challenge the accuracy of the previous studies. The New Tuxedo software suite was used for the main analyses. The results revealed novel, different sets of reference genes for each of the tissues with 17 common genes among the top 10 genes lists of 16 tissues. The results did disprove the suitability of WU-HKGs such as Actb, Ldha, Scd, B2m, and Hprt1 for any of the tissues examined. On the contrary, a total of 6, 13, 14, 23, and 32 validated housekeeping genes (V-HKGs) were discovered as the most stable and suitable reference genes for muscle, spleen, liver, heart, and kidney tissues, respectively. Although we identified a few new HKGs usable for multiple tissues, the selection of suitable HKGs is required to be tissue specific. The newly introduced reference genes from the present study, despite lacking experimental validation, will be able to contribute to the more accurate normalization for future expression analysis of chicken genes.

Identification of reference genes for expression studies in the liver, and spleen of laying hens housed in cage and cage-free systems

“…Transcriptome analysis using technologies such as DNA microarrays, RNA-Seq, and other methods is in demand to evaluate gene expression ( Lovén et al , 2012 ; Hasanpur et al , 2022 ). However, the qPCR remains a valid and preferred tool to validate the results obtained by transcriptomic analyses ( Garrido et al , 2020 ; Hasanpur et al , 2022 ).…”

“…Transcriptome analysis using technologies such as DNA microarrays, RNA-Seq, and other methods is in demand to evaluate gene expression ( Lovén et al , 2012 ; Hasanpur et al , 2022 ). However, the qPCR remains a valid and preferred tool to validate the results obtained by transcriptomic analyses ( Garrido et al , 2020 ; Hasanpur et al , 2022 ). Normalization strategies are needed to achieve accurate results, and reference genes as markers of stability is a common and effective method used for qPCR normalization in the relative quantification method ( Vandesompele et al , 2002 ; Samiullah et al , 2017 ; Garrido et al , 2020 ).…”

“…Several studies have been conducted on screening reference genes in various tissues of laying hens and geese ( Nascimento et al , 2015 ; Zhang et al , 2021 ). For example, using next-generation sequencing data, Hasanpur et al (2022) report the Ap2m1 gene as a suitable reference gene for the liver and the Rpl6 gene as a relatively appropriate reference gene for the spleen in chicken. In contrast, Mogilicherla et al (2022) reported the ALB gene in the liver and the GAPDH gene in the spleen as the most stable reference genes in Gallus gallus.…”

Screening of reliable reference genes for the normalization of RT-qPCR in chicken gastrointestinal tract

“…The optimal reference genes are characterized by their consistent expression across various tissue types and cells, unaffected by external factors such as the environment, experimental conditions, or other variables. Nevertheless, the absence of a universally ideal reference gene applicable to all tissues necessitates caution ( Hasanpur et al, 2022 ; Mogilicherla et al, 2022 ), as certain commonly utilized reference genes, including glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), beta-actin ( β-Actin ), exhibit unsuitability in specific tissues due to their inherent variability ( Glare et al, 2002 ; Barber et al, 2005 ; Lin and Redies, 2012 ).…”

“…In recent years, there has been a significant increase in scholarly investigations concerning reference genes in various tissues of domesticated animals ( Bonnet et al, 2013 ; Vorachek et al, 2013 ; Cieslak et al, 2015 ; Park et al, 2015 ; Faheem and Khaliq, 2019 ; Wang et al, 2022 ). Notably, in the case of chickens ( Gallus gallus ), several studies have successfully identified stable reference genes under different conditions ( Cedraz De Oliveira et al, 2017 ; Hassanpour et al, 2019 ; Rodríguez Hernández et al, 2021 ), and the evaluation of genes employed for gene expression analysis in major chicken organs has also been established ( Hasanpur et al, 2022 ). However, the absence of a dependable reference gene applicable to multiple tissues remains a challenge.…”