Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos

Stowe, Heather M.; Curry, Erin; Calcatera, Samantha M.; Krisher, Rebecca L.; Paczkowski, Melissa; Pratt, Scott L.

doi:10.1016/j.gene.2012.03.058

Cited by 35 publications

(24 citation statements)

References 49 publications

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“…Analyses of zebrafish Dicer1 during zebrafish development showed that Dicer1 is highly expressed in oocytes, early cleavage stage embryos, and at late stage of development. The results are consistent with the recent findings of porcine Dicer1 that was expressed during embryogenesis [19]. In the adult zebrafish, Dicer1 expression was observed in all the tissues examined.…”

Section: Discussionsupporting

confidence: 93%

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Akhtar

Patnaik

Raghupathy

et al. 2015

Journal of Ophthalmology

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DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA) and RNA-interference (RNAi) functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE) cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter) induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter) have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE) cells are normal with no sign of degeneration at the age of 20 months.

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Section: Discussionsupporting

confidence: 93%

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Akhtar

Patnaik

Raghupathy

et al. 2015

Journal of Ophthalmology

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“…Total cellular RNA (tcRNA) was isolated from testis and epididymis samples using the mirVana mRNA isolation kit (Ambion, Austin, Texas, USA) as previously reported [36]. The RNA purity was assessed using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA) to determine A260:A280 ratio.…”

Section: Reverse Transcription-polymerase Chain Reactionmentioning

confidence: 99%

Identification of bovine prolactin in seminal fluid, and expression and localization of the prolactin receptor and prolactin-inducible protein in the testis and epididymis of bulls exposed to ergot alkaloids

et al. 2015

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“…In this sense, only a few studies have explored the stability of some miRNAs in human tissues [19]–[21] and the published works are largely related to cancer processes [24]–[28]. Focusing on miRNA expression studies in livestock species, there are few works using miRNAs as reference control in miRNA expression analysis by relative quantification [29]–[31] and only one report has deeply analysed the miRNA expression stability in pigs to be used as reference miRNAs [22].…”

Section: Introductionmentioning

confidence: 99%

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

et al. 2012

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Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.

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Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos

Cited by 35 publications

References 49 publications

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Identification of bovine prolactin in seminal fluid, and expression and localization of the prolactin receptor and prolactin-inducible protein in the testis and epididymis of bulls exposed to ergot alkaloids

Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

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