Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material

El-Khoury, Victoria; Pierson, Sandrine; Kaoma, Tony; Bernardin, François; Berchem, Guy

doi:10.1038/srep19529

Cited by 145 publications

(135 citation statements)

References 33 publications

(67 reference statements)

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“…Furthermore, isolates from different methods were reproducibly enriched in fragments of additional short non-coding RNA classes such as tRNA. We have used two different RNA extraction kits in this study, and varying combinations of EV isolation and RNA extraction methods might yield slightly different results [5,52]. However, based on the vastly different characteristics of the material captured by each method, we believe that EV isolation itself has a far greater impact on downstream RNA analysis than the respective extraction method (Figure 6).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

191

165

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Extracellular vesicles (EVs) are intercellular communicators with key functions in physiological and pathological processes and have recently garnered interest because of their diagnostic and therapeutic potential. The past decade has brought about the development and commercialization of a wide array of methods to isolate EVs from serum. Which subpopulations of EVs are captured strongly depends on the isolation method, which in turn determines how suitable resulting samples are for various downstream applications. To help clinicians and scientists choose the most appropriate approach for their experiments, isolation methods need to be comparatively characterized. Few attempts have been made to comprehensively analyse vesicular microRNAs (miRNAs) in patient biofluids for biomarker studies. To address this discrepancy, we set out to benchmark the performance of several isolation principles for serum EVs in healthy individuals and critically ill patients. Here, we compared five different methods of EV isolation in combination with two RNA extraction methods regarding their suitability for biomarker discovery-focused miRNA sequencing as well as biological characteristics of captured vesicles. Our findings reveal striking method-specific differences in both the properties of isolated vesicles and the ability of associated miRNAs to serve in biomarker research. While isolation by precipitation and membrane affinity was highly suitable for miRNA-based biomarker discovery, methods based on size-exclusion chromatography failed to separate patients from healthy volunteers. Isolated vesicles differed in size, quantity, purity and composition, indicating that each method captured distinctive populations of EVs as well as additional contaminants. Even though the focus of this work was on transcriptomic profiling of EV-miRNAs, our insights also apply to additional areas of research. We provide guidance for navigating the multitude of EV isolation methods available today and help researchers and clinicians make an informed choice about which strategy to use for experiments involving critically ill patients.

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Section: Discussionmentioning

confidence: 99%

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Buschmann

Kirchner

Hermann

et al. 2018

J of Extracellular Vesicle

191

165

View full text Add to dashboard Cite

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“…For normalization purposes, 3.5 µl of a synthetic Caenorhabditis elegans miR-39 was spiked in after lysis before the preparation of the samples. RNA yields isolated from plasma were insufficient for proper quantitation with, for example, a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, Del., USA) [28]. Therefore, as recommended in the literature in this setting [18], equal volumes of RNA, rather than equal amounts of RNA, were used as input in the reverse transcription reaction.…”

Section: Methodsmentioning

confidence: 99%

Temporal Profile of Circulating microRNAs after Global Hypoxia-Ischemia in Newborn Piglets

Garberg¹,

Huun²,

Baumbusch³

et al. 2016

Neonatology

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Background: There is a lack of reliable biomarkers that can identify and grade acute hypoxic-ischemic encephalopathy in newborns. MicroRNAs (miRNA) are short, non-coding strands of RNA that are released into the circulation in response to tissue stress and injury. Some miRNAs are highly tissue specific and thus may potentially be non-invasive biomarkers of neonatal hypoxic-ischemic brain injury. Objective: The aim of this study was to characterize the temporal expression of selected circulating miRNAs in a clinically relevant piglet model of neonatal hypoxia-ischemia (HI). Methods: A total of 13 anesthetized newborn piglets were randomized to either a control group (n = 5) or transient global HI group (n = 8). HI was achieved by ventilation with 8% oxygen until the point of severe acidosis (arterial base excess ≤-20 mmol/l) and/or hypotension (mean arterial blood pressure ≤20 mm Hg) was reached. Plasma was sampled at baseline, at the end of HI and 0.5, 3.5 and 9.5 h after HI. MiRNA expression was measured by qRT-PCR. Results: Compared to baseline, miR-374a increased during HI (p = 0.01), remained elevated at 0.5 h after HI (p = 0.02) and was downregulated at 9.5 h after HI (p = 0.02). MiR-210 increased during HI (p = 0.02) and rapidly normalized by 0.5 h after HI. MiR-124 and miR-125b did not exhibit significant alterations. Correlations were observed between miR-374a, arterial pH, base excess and lactate levels, and between miR-210 and pO₂ (p < 0.05). Conclusions: Our data suggest that miR-374a and miR-210 are important regulators in neonatal HI and might have a place as biomarkers in this setting.

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“…We purified total RNA from testis and injected a quantity corresponding to the amount found in one spermatid (50 pg) to embryos generated with sperm. TRIzol was used to isolate RNA as it allows the recovery of RNA in a broad range of sizes (El-Khoury et al 2016). All embryos generated in that way developed normally, indicating that testicular RNAs are not detrimental to embryonic development (Supplemental Fig.…”

Section: Developmentally Important Genes Are Misregulated In Spermatimentioning

confidence: 99%

Sperm is epigenetically programmed to regulate gene transcription in embryos

et al. 2016

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For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.

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Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material

Cited by 145 publications

References 33 publications

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

Temporal Profile of Circulating microRNAs after Global Hypoxia-Ischemia in Newborn Piglets

Sperm is epigenetically programmed to regulate gene transcription in embryos

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