Identification of reference micro<scp>RNA</scp>s for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic–polycytidylic acid

Wang, H.; Wang, J.; Sun, Stella; Wang, Y.; Guo, Jianfeng; Ning, Chao; Yang, Ke; Liu, J. F.

doi:10.1111/iji.12198

Cited by 6 publications

(3 citation statements)

References 46 publications

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“…A total of 4 μl of diluted template cDNA was used per 10 μl reaction. U6 SNRNA, a small nuclear RNA, was used as a reference gene as it is frequently utilised for studies of miRNA expression such as this [ 127 , 128 ]. UniSP6 was spiked in during cDNA synthesis, acting as an internal control.…”

Section: Methodsmentioning

confidence: 99%

Temporally Altered miRNA Expression in a Piglet Model of Hypoxic Ischemic Brain Injury

et al. 2020

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Hypoxic ischemic encephalopathy (HIE) is the most frequent cause of acquired infant brain injury. Early, clinically relevant biomarkers are required to allow timely application of therapeutic interventions. We previously reported early alterations in several microRNAs (miRNA) in umbilical cord blood at birth in infants with HIE. However, the exact timing of these alterations is unknown. Here, we report serial changes in six circulating, cross-species/bridging biomarkers in a clinically relevant porcine model of neonatal HIE with functional analysis. Six miRNAs—miR-374a, miR-181b, miR-181a, miR-151a, miR-148a and miR-128—were significantly and rapidly upregulated 1-h post-HI. Changes in miR-374a, miR-181b and miR-181a appeared specific to moderate-severe HI. Histopathological injury and five miRNAs displayed positive correlations and were predictive of MRS Lac/Cr ratios. Bioinformatic analysis identified that components of the bone morphogenic protein (BMP) family may be targets of miR-181a. Inhibition of miR-181a increased neurite length in both SH-SY5Y cells at 1 DIV (days in vitro) and in primary cultures of rat neuronal midbrain at 3 DIV. In agreement, inhibition of miR-181a increased expression of BMPR2 in differentiating SH-SY5Y cells. These miRNAs may therefore act as early biomarkers of HIE, thereby allowing for rapid diagnosis and timely therapeutic intervention and may regulate expression of signalling pathways vital to neuronal survival. Electronic supplementary material The online version of this article (10.1007/s12035-020-02018-w) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Temporally Altered miRNA Expression in a Piglet Model of Hypoxic Ischemic Brain Injury

et al. 2020

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“…In order to assess the reliability of the identified miRNAs, nine miRNAs representing different expression levels were chosen to be validated by quantitative real-time PCR (qPCR) using the miScript PCR System (Qiagen, Hilden, Germany). Ssc-miR-34a and Ssc-miR-107 were selected as endogenous controls, which were confirmed to be stably expressed in the PBMC [24]. For each of the nine miRNAs tested and two endogenous controls, one miRNA-specific primer was designed, as detailed in Table S14.…”

Section: Resultsmentioning

confidence: 99%

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs

Wang

et al. 2016

IJMS

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MicroRNAs (miRNAs) are one family of small noncoding RNAs that function to modulate the activity of specific mRNA targets in animals. To understand the role of miRNAs in regulating genes involved in the host immune response to RNA viruses, we profiled and characterized the miRNAs of swine peripheral blood mononuclear cells (PBMC) stimulated with poly I:C, a synthetic dsRNA analog, by miRNA-sequencing (miRNA-seq). We identified a total of 905 miRNAs, of which 503 miRNAs were firstly exploited herein with no annotation in the latest miRBase 21.0. Expression analysis demonstrated that poly I:C stimulation can elicit significantly differentially expressed (DE) miRNAs in Dapulian (n = 20), one Chinese indigenous breed, as well as Landrace (n = 23). By integrating the mRNA expression profiles of the same sample with miRNA profiles, we carried out function analyses of the target genes of these DE miRNAs, with the results indicating that target genes were most enriched in some immune-related pathways and gene ontology (GO) terms, suggesting that DE miRNAs play an important role in the regulation of host to poly I:C stimulation. Furthermore, we also detected 43 and 61 significantly DE miRNAs between the two breeds in the control sample groups and poly I:C stimulation groups, respectively, which may be involved in regulation of the different characteristics of the two breeds. This study describes for the first time the PBMC miRNA transcriptomic response to poly I:C stimulation in pigs, which not only contributes to a broad view of the pig miRNAome but improves our understanding of miRNA function in regulating host immune response to RNA viruses.

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“…However, despite validation, the positive control has no significant influence on the stability evaluation by the calculation software. Thus, many studies [14, 36–39] evaluated suitable reference genes by calculation software without the positive control.…”

Section: Discussionmentioning

confidence: 99%

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Diao

Zhao

et al. 2017

BMC Molecular Biol

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BackgroundOxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).ResultsUsing geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

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Identification of reference microRNAs for quantitative expression analysis in porcine peripheral blood mononuclear cells treated with polyinosinic–polycytidylic acid

Cited by 6 publications

References 46 publications

Temporally Altered miRNA Expression in a Piglet Model of Hypoxic Ischemic Brain Injury

Temporally Altered miRNA Expression in a Piglet Model of Hypoxic Ischemic Brain Injury

MicroRNA Transcriptome of Poly I:C-Stimulated Peripheral Blood Mononuclear Cells Reveals Evidence for MicroRNAs in Regulating Host Response to RNA Viruses in Pigs

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

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