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. 2022 Nov 2;12(1):18473.
doi: 10.1038/s41598-022-22588-0.

Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh

Manish Tiwari  1 , Monika Sodhi  1 , Preeti Verma  1 , Prince Vivek  1 , Ranjit S Kataria  1 , Saket K Niranjan  1 , Vijay K Bharti  2 , Nampher Masharing  1 , Gayatri Gujar  1 , Divya Chanda  1 , Manishi Mukesh  3
Affiliations

Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh

Manish Tiwari et al. Sci Rep. .

Abstract

The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat, Double hump cattle and Zanskar ponies were included in this study. The commonly used statistical algorithms such as geNorm, Normfinder, BestKeeper and RefFinder were employed to assess the stability of these RGs in all the livestock species. Our study has identified different panel of reference genes in each species; for example, EEF1A1, RPL4 in Ladakhi cattle; GAPDH, RPS9, ACTB in Ladakhi yak; HPRT1, B2M, ACTB in Ladakhi donkey; HPRT1, B2M, ACTB in Double hump camel, RPS9, HPRT1 in Changthangi goat, HPRT1 and ACTB in Zanskar ponies. To the best of our knowledge, this is the first systematic attempt to identify panel of RGs across different livestock species types adapted to high altitude hypoxia conditions. In future, the findings of the present study would be quite helpful in conducting any transcriptional studies to understand the molecular basis of high altitude adaptation of native livestock population of Leh-Ladakh.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression level of individual candidate RGs in LAC (a), LAY (b), LAD (c), CHG (d), DHC (e) and ZAP (f). The data is presented as quantification cycle (Ct) values of each gene in the box-whisker diagram. The median is shown as a line across the box while whiskers indicate maximum and minimum values.
Figure 2
Figure 2
geNorm analysis for ranking of genes based on average expression stability measure (M value), Pair-wise variation (Vn/Vn + 1) between the normalization factors NFn and NFn + 1 to determine the optimal number of reference genes and Normfinder analysis in LAC (a–c), LAY (d–f), LAD (g–i), CHG (j–l), DHC (m–o) and ZAP (p–r respectively).
Figure 3
Figure 3
The entire workflow of the qPCR experiment conducted in PBMCs of different species adapted to cold arid hypoxia environment. Maps was made using Mapchart (https://www.mapchart.net/india.html).
See this image and copyright information in PMC

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