MicroRNA isolation and stability in stored RNA samples

Mráz, Marek; Malinová, Karla; Mayer, Jiřı́; Pospı́šilová, Šárka

doi:10.1016/j.bbrc.2009.09.061

Cited by 197 publications

(139 citation statements)

References 25 publications

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“…In contrast to other RNA molecules, the circulating miRs are stable and consistent across individuals of the same species, 45 and recent reports also indicate that mature miRs are highly stable in circulation. 12,46 Most of the published studies that have examined circulating miRs as biomarkers have focused on cancer, 47 but a few reports studied the role of circulating miRs in non-neoplastic diseases. Our research showed that the change in miR-206 expression in the serum in the hypoxic PH rat model was likely to be time-dependent.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Yue

Guan

Wang

et al. 2013

Laboratory Investigation

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Hypoxia-induced pulmonary hypertension (PH), which is characterized by vasoconstriction and subsequent structural remodeling of blood vessels, is an important event in chronic obstructive pulmonary disease patients and in people living at high altitudes. Hypoxia-inducible factor-1a (HIF-1a) and its regulator four-and-a-half LIM (Lin-11, Isl-1 and Mec-3) domain 1 (Fhl-1) have important roles in hypoxia-induced PH. is critical for myogenesis and related diseases; however, the role of miR-206 in hypoxia-induced PH is unknown. miR-206 expression was evaluated in a hypoxic rat model and in cultured hypoxic pulmonary artery smooth muscle cells (PASMCs) using real-time quantitative PCR (RT-qPCR). HIF-1a and Fhl-1 expression were evaluated using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence. The function of miR-206 was assessed by transfecting miR-206 mimics and inhibitors. Dualluciferase reporter gene assays and western blotting were performed to validate the target genes of miR-206. siRNA targeted against Fhl-1 was used to investigate the effect of Fhl-1 on miR-206. Flow cytometry was used to detect the cell cycle phase distribution in each group of PASMCs. Significant downregulation of miR-206 in hypoxic lung tissue and PASMCs was identified, whereas HIF-1a and Fhl-1 were upregulated in these samples. The expression of miR-206 in the serum was different from that in the lung tissue. Transfection of pre-miR miR-206 in hypoxic conditions led to increased expression of HIF-1a and Fhl-1 rather than abolishing hypoxia-induced HIF-1a and Fhl-1, as was expected, and promoted the entry of cells into the S phase and enhanced PASMC proliferation. Fhl-1-targeted siRNA in PASMC prevented cell proliferation and led to an increased proportion of cells in the G1 phase without altering miR-206 expression. Bioinformatic analysis and dual-luciferase reporter gene assays revealed direct evidence for miR-206 targeting of HIF-1a. In conclusion, hypoxia-induced downregulation of miR-206 promotes PH by targeting the HIF-1a/Fhl-1 pathway in PASMCs. miR-206 could be a triggering factor of early stage of hypoxia-induced PH. Hypoxia-induced pulmonary hypertension (PH) is a serious disorder characterized by pulmonary vasoconstriction and enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) that lead to structural remodeling of blood vessel walls. PH is a common clinical manifestation of chronic obstructive pulmonary disease (COPD) and in people who live at high altitude. 1 Hypoxia-inducible factor-1a (HIF-1a) has a key role in the pathophysiology of hypoxia-induced PH.The four-and-a-half LIM domain 1 (Fhl-1) protein, a member of the Fhl family, has also been implicated in the biological pathways of muscle growth and differentiation in PH 2-4 and is regulated by HIF-1a in a feedback loop that serves to limit HIF-1a activity under conditions of prolonged hypoxia. 5 MicroRNAs (miRs) are a class of B23-nucleotide, noncoding single-stranded RNAs that regulate gene function by identifying the 3 0 -untransl...

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Section: Discussionmentioning

confidence: 99%

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Yue

Guan

Wang

et al. 2013

Laboratory Investigation

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“…Bravo et al (40 ) observed high instability of miRNAs and their cDNA derivatives stored at Ϫ80°C as soon as 3 days after RNA isolation; however, 2 teams who reevaluated this result with the same isolation methods and storage conditions could not confirm this instability (38,39 ). The necessity to maintain strict ribonuclease-free conditions in all miRNApreparation steps has been pointed out (38 ). Under these conditions, RT-qPCR analyses of miRNAs showed stable data for RNA samples stored at Ϫ80°C over a period of 10 months (38 ).…”

Section: Chived In Clinical Diagnosis Is Heavily Degraded (28 )mentioning

confidence: 99%

Robust MicroRNA Stability in Degraded RNA Preparations from Human Tissue and Cell Samples

et al. 2010

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BACKGROUND: RNA integrity is the essential factor that determines the accuracy of mRNA transcript measurements obtained with quantitative real-time reversetranscription PCR (RT-qPCR), but evidence is clearly lacking on whether this conclusion also applies to microRNAs (miRNAs). We evaluated this issue by comparative analysis of the dependence of miRNA and mRNA measurements on RNA integrity in renal and prostate samples, under both model and clinical conditions.

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“…RNA was isolated using TRIzol® (Gibco, Invitrogen, Carlsbad, CA, USA) [19]. One microgram of RNA was respectively reverse transcribed (iScript® cDNA synthesis Kit, Bio-Rad) to cDNA.…”

Section: Controlsmentioning

confidence: 99%

SOCS3 and SOCS5 mRNA expressions may predict initial steroid response in nephrotic syndrome children

Ostalska‐Nowicka

Śmiech²,

Jaroniec³

et al. 2012

Folia Histochem Cytobiol.

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Suppressors of Cytokine Signaling (SOCS) inhibit Signal Transducers and Activators of Transcription (STATs) phosphorylation by binding and inhibiting Janus Kinases (JaKs). The aim of the present study was to evaluate the influence of glucocorticosteroids on the JaK/STAT signaling pathway in the leukocytes of nephrotic syndrome (NS) patients. The study group was composed of 34 steroid sensitive NS (SSNS) children and 20 steroid resistant NS (SRNS) subjects. Gene expression was assessed by real-time PCR using pre-designed human JaK/STAT PCR array. Protein expression was evaluated using ELISA assay (plasma concentration) and immunofluorescence (in situ protein expression). In SSNS children, the initial increased expression of JaK1, JaK2, JaK3, STAT1, STAT2, STAT6, TYK2, SOCS1, SOCS2, SOCS3, SOCS4 and SOCS5 was reduced back to the control limits. Similarly, in SRNS patients the increased levels of almost all mRNA expressions for the abovementioned genes were decreased, with the exceptions of SOCS3 and SOCS5 expressions. These mRNA expressions were still significantly increased and correlated with early unfavorable course of nephrotic syndrome in children. Plasma levels of SOCS3, SOCS5, IL-6 and IL-20 were significantly increased in SRNS subjects after six weeks of steroids medication compared to SSNS and control participants. We conclude that SOCS3 and SOCS5 increased mRNA expressions might predict initial resistance

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MicroRNA isolation and stability in stored RNA samples

Cited by 197 publications

References 25 publications

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Robust MicroRNA Stability in Degraded RNA Preparations from Human Tissue and Cell Samples

SOCS3 and SOCS5 mRNA expressions may predict initial steroid response in nephrotic syndrome children

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