How to Choose a Normalization Strategy for Mirna Quantitative Real-Time (Qpcr) Arrays

Deo, Ameya; Carlsson, Jessica; Lindlöf, Angelica

doi:10.1142/s0219720011005793

Cited by 52 publications

(52 citation statements)

References 37 publications

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“…For each miRNA, all reactions were run in triplicate. The relative amount of each miRNA to U6 snRNA was evaluated using the equation 2 -ΔΔCT according to the threshold cycle (CT) (Deo et al, 2011).…”

Section: Confirmation Of Differentially Expressed Mirnas Using Real-tmentioning

confidence: 99%

Characterization and expression analysis of microRNAs in Qira black sheep and Hetian sheep ovaries using Solexa sequencing

Shen¹,

Chen²,

Jia³

et al. 2015

Genet. Mol. Res.

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ABSTRACT. The role of microRNAs (miRNAs) in the regulation of mammalian reproduction has been demonstrated previously. However, only a few studies have assessed the role of miRNAs in the reproduction processes of sheep. The elucidation of miRNA expression profiles in the ovaries of different sheep breeds representing fecundity extremes will be useful in understanding the roles of miRNAs in sheep reproduction. In this study, two small RNA libraries were constructed from ovary tissue taken from Qira black sheep and Hetian sheep during the estrous period and then sequenced using the Solexa sequencing method. We obtained 9,565,212 and 9,563,426 high-quality reads from Qira black sheep and Hetian sheep, respectively. In total, 531 miRNAs, including 98 putative miRNAs, were identified. Among the conserved miRNAs, 125 known miRNAs were significantly differentially expressed in the Qira black sheep and Hetian sheep libraries, with 24 upregulated and 101 downregulated in the Hetian sheep compared to the Qira black sheep. Four differentially expressed miRNAs were analyzed using real-time 7357 miRNA expression analysis in sheep ovaries ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 7356-7367 (2015) quantitative PCR to validate the reliability of the Solexa sequencing results. These results provide a foundation for future research on the regulation of miRNAs in sheep fertility and enrich the sheep miRNA databases.

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Section: Confirmation Of Differentially Expressed Mirnas Using Real-tmentioning

confidence: 99%

Characterization and expression analysis of microRNAs in Qira black sheep and Hetian sheep ovaries using Solexa sequencing

Shen¹,

Chen²,

Jia³

et al. 2015

Genet. Mol. Res.

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“…Each sample from each individual animal was analyzed in triplicate. Normalized factors of the internal control gene (U6 snRNA) and the relative quantities of the miRNAs were analyzed using the qBase software (Deo et al, 2011 …”

Section: Real-time Quantitative Pcr (Q-pcr)mentioning

confidence: 99%

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology

Ling

Ding²,

Zhang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 speciesconserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of realtime quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

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“…Negative controls were also run in each set of PCR assays, including without complementary DNA (cDNA) (no template controls, NTCs) and without reverse transcriptase. The fold expression or repression of the target gene relative to the internal control gene GAPDH (Tu et al 2015) in each sample was then calculated by the 2 -ΔΔCt method (Deo et al 2011). …”

Section: Methodsmentioning

confidence: 99%

Dynamic expression of HSP90B1 mRNA in the hypothalamus of two Chinese chicken breeds under heat stress and association analysis with a SNP in Huainan chickens

Wan¹,

Ma²,

Wei³

et al. 2017

CZECH J ANIM SCI

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Wan Y., Ma C., Wei P., Fang Q., Guo X., Zhou B., Jiang R. (2017): Dynamic expression of HSP90B1 mRNA in the hypothalamus of two Chinese chicken breeds under heat stress and association analysis with a SNP in Huainan chickens. Czech J. Anim. Sci., 62, 82-87.The effects of heat stress on HSP90B1 messenger RNA (mRNA) expression in the hypothalamus of chicken were investigated and HSP90B1 variations were detected. Females of two Chinese chicken breeds (Huainan and Wenchang) were used for the experiments. At 64 days of age, the ambient temperature (24 ± 1°C) was increased to 35 ± 1°C for 24 h (heat stress), then decreased to 24 ± 1°C for 24 h (recovery). Hypothalamus samples were collected at 0, 12, and 24 h during heat stress, as well as 12 and 24 h during recovery. The HSP90B1 mRNA expression increased significantly during heat stress and significantly decreased during recovery being higher in Huainan chickens. Fifteen primer pairs were designed to amplify the exons of HSP90B1 by a polymerase chain reaction, and single nucleotide polymorphisms (SNPs) were detected by Sanger sequencing. In Huainan chickens, we identified a SNP (NC_006088.3:g.6798G>A) in exon 14 of HSP90B1 which did not cause amino acid variation but caused a codon for glutamic acid change from GAG to GAA. The frequencies for genotypes AA, GA, and GG were 0.49, 0.27, and 0.24, respectively. Individuals with the GG genotype survived heat stress at 42°C for a longer time (248.2 ± 39.3 min) than individuals with GA and AA genotypes, which survived for 227.2 ± 44.5 min and 179.3 ± 36.5 min, respectively. The results suggested that the increased heat tolerance was associated with the higher expression of HSP90B1, and genotype GG could be used as a potential marker for heat resistance in chickens.

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How to Choose a Normalization Strategy for Mirna Quantitative Real-Time (Qpcr) Arrays

Cited by 52 publications

References 37 publications

Characterization and expression analysis of microRNAs in Qira black sheep and Hetian sheep ovaries using Solexa sequencing

Characterization and expression analysis of microRNAs in Qira black sheep and Hetian sheep ovaries using Solexa sequencing

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology

Dynamic expression of HSP90B1 mRNA in the hypothalamus of two Chinese chicken breeds under heat stress and association analysis with a SNP in Huainan chickens

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