Hasanpur Karim, Hosseinzadeh Sevda, Mirzaaghayi Atiye, Alijani Sadegh
Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Front Genet. 2022 Sep 13;13:827538. doi: 10.3389/fgene.2022.827538. eCollection 2022.
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 , , , , and 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.
使用管家基因（HKGs）对基因表达分析进行准确标准化至关重要。为此，为特定实验选择一组合适的HKGs非常重要。尽管有许多研究，但对于用于鸡组织定量实时PCR分析的合适HKG集尚无共识。少数HKGs已被广泛使用。然而，对所有组织广泛使用少数HKGs具有挑战性。高通量基因表达RNA-seq数据的出现使得能够同时比较多个HKGs的稳定性。因此，我们利用每个组织至少三个数据集的平均变异系数，对所有可靠表达的基因（REGs；在至少一个样本中FPKM≥1）进行排序，并为16种鸡组织中的每一种引入了前10个最合适和最稳定的参考基因。我们使用相同的方法在其他数据集上评估了五个组织结果的一致性。此外，我们评估了96个先前广泛使用的HKGs（WU-HKGs），以检验先前研究的准确性。主要分析使用了New Tuxedo软件套件。结果显示，每个组织都有新的、不同的参考基因集，16个组织的前10个基因列表中有17个共同基因。结果确实证明了WU-HKGs如 、 、 、 和 不适用于所检测的任何组织。相反，分别发现了总共6个、13个、14个、23个和32个经过验证的管家基因（V-HKGs），作为肌肉、脾脏、肝脏、心脏和肾脏组织最稳定和合适的参考基因。虽然我们鉴定出了一些可用于多个组织的新HKGs，但合适的HKGs选择需要针对特定组织。本研究新引入的参考基因，尽管缺乏实验验证，但将有助于对鸡基因未来的表达分析进行更准确的标准化。
