Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection

Sahu, Anamika; Wani, Sajad A.; Saxena, Shikha; Rajak, Kaushal Kishor; Chaudhary, Dheeraj; Sahoo, Aditya Prasad; Khanduri, Alok; Pandey, Awadh Bihari; Mondal, Piyali; Malla, Waseem Akram; Khan, Raja Ishaq Nabi; Tiwari, Ashok Kumar; Mishra, Bina; Muthuchelvan, D.; Mishra, Bishnu Prasad; Singh, Raj Kumar; Gandham, Ravi Kumar

doi:10.1038/s41598-018-34236-7

Cited by 23 publications

(21 citation statements)

References 66 publications

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“…Both algorithms produced consistent results, suggesting that Normfinder and geNorm are robust programs for finding HK genes. In fact, those two programs have been used widely to determine HK genes in human, animals, and plants (Czechowski et al, 2005;Jian et al, 2008;Jongsik et al, 2007;Sahu et al, 2018). The stability value of our candidate HK genes were much lower than those in plants (Czechowski et al, 2005), suggesting that the candidate genes were stably expressed.…”

Section: Discussionmentioning

confidence: 91%

“…HK genes are often used as endogenous references to normalize the variations in RT-qPCR experiments (Jian et al, 2008). However, under conditions such as heat stress, viral infection, and cancer development, the expression of HK genes of animals can vary (Sahu et al, 2018). The utilization of invalidated reference genes results in unreliable and misleading conclusions.…”

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confidence: 99%

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Identification of Suitable Meloidogyne spp. Housekeeping Genes

DiGennaro

2019

Journal of Nematology

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Gene expression studies often require reliable housekeeping (HK) genes to accurately capture gene expression levels under given conditions. This is especially true for root-knot nematodes (RKN, Meloidogyne spp.), whose drastic developmental changes are strongly dependent upon their environment. Here we utilized a publicly available M. hapla RNAseq database to identify putative HK genes throughout the nematode lifecycle. We then validated these candidate HK genes on M. incognita in order to develop a small library of suitable HK genes for RKN. Seven putative HK genes were selected for validation based on high expression level and ease of primer design. The expression of these genes was quantified by qPCR at different developmental stages to capture the entire life cycle of M. incognita which included eggs and naive infective juveniles through 3-wk post inoculation. Two algorithms, geNorm and Normfinder, identified three genes (Disu, Poly, and Skinase) constitutively and uniformly expressed throughout the entire life cycle of RKN. We believe these genes are superior HK genes suitable to be used as internal reference genes at all stages of RKN. Importantly, while we identified Actin, a commonly used HK gene, as a candidate gene within our RNAseq analyses, our qPCR results did not demonstrate stable expression throughout the nematode life cycle of this gene. This study successfully validated suitable HK genes utilizing both RNAseq data and standard qPCR methods across two species of RKN; suitable HK genes are likely applicable to other species of RKN, or even plant-parasitic nematodes. Additional lists of potential HK genes are also provided if the nematode of interest does not have homologues of the three superior reference genes described here. Gene expression studies on RKN should use validated HK genes to ensure accurate representation of transcript abundance.

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Section: Discussionmentioning

confidence: 91%

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confidence: 99%

Identification of Suitable Meloidogyne spp. Housekeeping Genes

DiGennaro

2019

Journal of Nematology

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“…The RNA samples prepared from tissues and cell lysates from unstimulated and in vitro stimulated whole blood were analyzed using a ProbeSet consisting of probes specific for 131 genes from Ovis aries . Additionally, nine reference genes were included based on published recommendations ( Zaros et al, 2010 ; Vorachek et al, 2013 ; Mahakapuge et al, 2016 ; Sahu et al, 2018 ). A complete list of genes and probe sequences is provided in Supplementary File , Probe Summary.…”

Section: Methodsmentioning

confidence: 99%

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes

Quirke

Maclean

Haack

et al. 2021

Journal of Animal Science

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Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R 2 = 0.3) as well as separation of tissues from pregnant and non-pregnant animals [lymph nodes (MANOVA P = 2.337e-09, R 2 = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R 2 = 0.47)]. Nine genes were differentially (FDR <0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (P < 0.05) by 24% in the reproductive tissue of the pregnant animals comparing to non-pregnant animals. Analysis of gene categories revealed that expression of genes of the T cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and non-pregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.

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“…In recent past, numerous studies have been conducted in similar lines to identify panel of appropriate RGs in several livestock species such as cattle [19,26] buffaloes [18,17], yak [27], pig [28], goat [29,30] sheep [31,30], horse [32] etc. These studies have represented wide array of environmental or experimental conditions such as responses to external stimuli (heat stress, endurance, exercise), physiological or developmental stages, lactation cycle, cellular response, etc.…”

Section: Introductionmentioning

confidence: 99%

Selection of Species Specific Panel of Reference Genes Across Native Livestock Species Adapted to Trans-Himalayan Region of Leh-ladakh

Tiwari

Sodhi

Verma

et al. 2021

Preprint

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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 (LAC), Ladakhi yak (LAY), Ladakhi donkey (LAD), Chanthangi goat (CHG), Double hump cattle (DHC) and Zanskar ponies (ZAP) 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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Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection

Cited by 23 publications

References 66 publications

Identification of Suitable Meloidogyne spp. Housekeeping Genes

Identification of Suitable Meloidogyne spp. Housekeeping Genes

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes

Selection of Species Specific Panel of Reference Genes Across Native Livestock Species Adapted to Trans-Himalayan Region of Leh-ladakh

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