Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)

Kaur, Rajvir; Sodhi, Monika; Sharma, Ankita; Sharma, Vishal; Verma, Preeti; Swami, Shelesh Kumar; Kumari, Parvesh; Mukesh, Manishi

doi:10.1371/journal.pone.0191558

Cited by 33 publications

(36 citation statements)

References 32 publications

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“…For mammary gland, arithmetic mean Cq of three housekeeping genes (HKG): Actb , Uxt and Rps9 , was used to calculate ΔCq (Cq Gene of interest —Cq Housekeeping genes ). Actb is a classically used HKG while Uxt and Rps9 are known to be very stable genes in lactating mammary gland [ 36 , 37 ]. For pituitary gland, arithmetic mean Cq of Actb , Vcp and Rps9 was used as HKG to calculate ΔCq.…”

Section: Methodsmentioning

confidence: 99%

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion

Sevrin

Boquien

Gandon

et al. 2020

Genes

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We previously demonstrated galactagogue effect of fenugreek in a rat model of lactation challenge, foreshadowing its use in women’s breastfeeding management. To assess longitudinal molecular mechanisms involved in milk synthesis/secretion in dams submitted to fenugreek supplementation, inguinal mammary, pituitary glands and plasma were isolated in forty-three rats nursing large 12 pups-litters and assigned to either a control (CTL) or a fenugreek-supplemented (FEN) diet during lactation. RT-PCR were performed at days 12 and 18 of lactation (L12 and L18) and the first day of involution (Inv1) to measure the relative expression of genes related to both milk synthesis and its regulation in the mammary gland and lactogenic hormones in the pituitary gland. Plasma hormone concentrations were measured by ELISA. FEN diet induced 2- to 3-times higher fold change in relative expression of several genes related to macronutrient synthesis (Fasn, Acaca, Fabp3, B4galt1, Lalba and Csn2) and energy metabolism (Cpt1a, Acads) and in IGF-1 receptor in mammary gland, mainly at L12. Pituitary oxytocin expression and plasma insulin concentration (+77.1%) were also significantly increased. Altogether, these findings suggest fenugreek might extend duration of peak milk synthesis through modulation of the insulin/GH/IGF-1 axis and increase milk ejection by activation of oxytocin secretion.

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Section: Methodsmentioning

confidence: 99%

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion

Sevrin

Boquien

Gandon

et al. 2020

Genes

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“…Primers were designed by Primer-BLAST ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) (Table 2 ). The ubiquitously expressed prefoldin-like chaperone ( UXT ) gene was used to normalize the expression level of the candidate gene [ 47 , 48 ]. Total RNA was transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China).…”

Section: Methodsmentioning

confidence: 99%

Comparative transcriptome analysis reveals that PCK1 is a potential gene affecting IMF deposition in buffalo

et al. 2020

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Background In China, although buffaloes are abundant, beef is mainly obtained from cattle, and this preference is mainly attributed to the low intramuscular fat (IMF) content of buffalo. Genetic factors are an important driver that affects IMF deposition. Results To reveal the intrinsic factors responsible for the low IMF content of buffalo, mRNA expression patterns in muscle and adipose tissue between buffalo and cattle were characterized by RNA sequencing analysis. The IMF content in Nanyang cattle was higher than that in Xinyang buffalo. A total of 1566 mRNAs expressed in adipose tissue showed differential expression between the longissimus dorsi muscles of buffalo and cattle. Functional annotation suggested a difference in the glycolysis/gluconeogenesis pathway between the two species. The results of RT-qPCR analysis and gain-of-function experiments confirmed the positive association between the IMF content and phosphoenolpyruvate carboxykinase 1 (PCK1) expression in buffalo. In both mouse C2C12 cells and cultured bovine myocytes, the activity of the PCK1 promoter in buffalo is lower than that in cattle. However, in mouse 3T3-L1 adipocytes and cultured bovine adipocytes, the activity of PCK1 in buffalo promoter is higher than that in cattle. Conclusions These results indicate the important role of PCK1 in buffalo IMF deposition and illustrate the differences between buffalo and cattle promoter activity that drive PCK1 expression. This research helps to establish a foundation for further studies investigating IMF deposition in buffalo.

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“…The pairwise correlation coefficient of all eleven tested reference genes indicates a high positive correlation. For normalization, we selected only the most stable reference genes as recommended previously [48,[60][61][62] .…”

Section: Reference Gene Validationmentioning

confidence: 99%

Catalogue of stage-specific transcripts in Ixodes ricinus and their potential functions during the tick life-cycle

et al. 2020

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Background: The castor bean tick Ixodes ricinus is an important vector of several clinically important diseases, whose prevalence increases with accelerating global climate changes. Characterization of a tick life-cycle is thus of great importance. However, researchers mainly focus on specific organs of fed life stages, while early development of this tick species is largely neglected. Methods: In an attempt to better understand the life-cycle of this widespread arthropod parasite, we sequenced the transcriptomes of four life stages (egg, larva, nymph and adult female), including unfed and partially blood-fed individuals. To enable a more reliable identification of transcripts and their comparison in all five transcriptome libraries, we validated an improved-fit set of five I. ricinus-specific reference genes for internal standard normalization of our transcriptomes. Then, we mapped biological functions to transcripts identified in different life stages (clusters) to elucidate life stage-specific processes. Finally, we drew conclusions from the functional enrichment of these clusters specifically assigned to each transcriptome, also in the context of recently published transcriptomic studies in ticks. Results: We found that reproduction-related transcripts are present in both fed nymphs and fed females, underlining the poorly documented importance of ovaries as moulting regulators in ticks. Additionally, we identified transposase transcripts in tick eggs suggesting elevated transposition during embryogenesis, co-activated with factors driving developmental regulation of gene expression. Our findings also highlight the importance of the regulation of energetic metabolism in tick eggs during embryonic development and glutamate metabolism in nymphs. Conclusions: Our study presents novel insights into stage-specific transcriptomes of I. ricinus and extends the current knowledge of this medically important pathogen, especially in the early phases of its development.

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Selection of suitable reference genes for normalization of quantitative RT-PCR (RT-qPCR) expression data across twelve tissues of riverine buffaloes (Bubalus bubalis)

Cited by 33 publications

References 32 publications

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion

Fenugreek Stimulates the Expression of Genes Involved in Milk Synthesis and Milk Flow through Modulation of Insulin/GH/IGF-1 Axis and Oxytocin Secretion

Comparative transcriptome analysis reveals that PCK1 is a potential gene affecting IMF deposition in buffalo

Catalogue of stage-specific transcripts in Ixodes ricinus and their potential functions during the tick life-cycle

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