Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Nov 10;9(11):943.
doi: 10.3390/ani9110943.

The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Xiaoyun Wu  1 , Xuelan Zhou  1 , Xuezhi Ding  1 , Min Chu  1 , Chunnian Liang  1 , Jie Pei  1 , Lin Xiong  1 , Pengjia Bao  1 , Xian Guo  1 , Ping Yan  1
Affiliations

The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Xiaoyun Wu et al. Animals (Basel). .

Abstract

Investigating the critical genes related to milk synthesis is essential for the improvement of the milk yield of the yak. Real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable and widely used method to measure and evaluate gene expression levels. Selection of suitable reference genes is mandatory to acquire accurate normalization of gene expression results from RT-qPCR. To select the most stable reference genes for reliable normalization of mRNA expression by RT-qPCR in the mammary gland of the Ashidan yak, we selected 16 candidate reference genes and analyzed their expression stability at different physiological stages (lactation and dry period). The expression stability of the candidate reference genes was assessed using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder methods. The results showed that the hydroxymethylbilane synthase gene (HMBS) and the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) were the most stable genes across all treatment samples. The reliability of selected reference genes was validated by normalizing relative expression of the lactation-related 60S ribosomal protein L35 gene (RPL35). The relative expression of RPL35 varied considerably according to the different reference genes. This work provides valuable information to further promote research in the molecular mechanisms involved in lactation and mammary gland development and provides a foundation for the improvement of the milk yield and quality of the Ashidan yak.

Keywords: RT-qPCR; mammary gland; reference gene.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cq values (expression levels) of 16 candidate reference genes in all tested samples. Each box indicates the 25th and 75th percentiles. Whisker caps correspond to the maximum and minimum values. A line within the boxes depicts the median.
Figure 2
Figure 2
Pairwise variation (V) analyses in all tested samples. The V cutoff value was set to 0.15, below which the inclusion of an additional reference gene would not have a beneficial effect on the normalization.
Figure 3
Figure 3
The normalized expression of the RPL35 mRNA in yak mammary gland at lactation and dry period. The expression levels were normalized using HMBS, YWHAZ or GAPDH as reference genes individually and with the mean of HMBS+YWHAZ. Values are expressed as mean ± SE. * p < 0.05.
See this image and copyright information in PMC

References

    1. Wiener G., Han J., Long R. The Yak. 2nd ed. FAO Regional office for Asia and the Pacific; Bangkok, Thailand: 2003. pp. 1–7.
    1. Ding L., Wang Y., Kreuzer M., Guo X., Mi J., Gou Y., Shang Z., Zhang Y., Zhou J., Wang H., et al. Seasonal variations in the fatty acid profile of milk from yaks grazing on the Qinghai-Tibetan plateau. J. Dairy Res. 2013;80:410–417. doi: 10.1017/S0022029913000496. - DOI - PubMed
    1. Kandeepan G., Sangma S. Optimization of the level of guar gum in low fat yak milk paneer. J. Stored. Prod. Res. 2010;1:9–12.
    1. Li H., Ma Y., Li Q., Wang J., Cheng J., Xue J., Shi J. The chemical composition and nitrogen distribution of Chinese yak (Maiwa) milk. Int. J. Mol. Sci. 2011;12:4885–4895. doi: 10.3390/ijms12084885. - DOI - PMC - PubMed
    1. New Yak Breed Brings Herders Renewed Hope. [(accessed on 8 July 2019)]; Available online: https://www.chinadaily.com.cn /a/201907/08/WS5d229d05a3105895c2e7c2d4.html.

LinkOut - more resources