Variability in microRNA recovery from plasma: Comparison of five commercial kits

Brunet-Vega, Anna; Pericay, Carles; Quilez, María Elisa; Ramírez‐Lázaro, María José; Calvet, Xavier; Lario, Sergio

doi:10.1016/j.ab.2015.07.018

Cited by 64 publications

(54 citation statements)

References 28 publications

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“…This result is likely due to the absence of pre-analytical considerations that are important to take into account before miRNA screening, e.g. ; serum vs plasma [71], methods used for RNA isolation [72], methods for miRNA screening (microarray, Low density array, sequencing) [73], technologies for validation by qRT-PCR [74] and data normalization [75]. Particularly, the circulating levels of some miRNAs are affected by hemolysis [53,76].…”

Section: Discussionmentioning

confidence: 99%

Diagnostic Value of Cell-free Circulating Micrornas for Obesity and Type 2 Diabetes: A Meta-analysis

Villard¹,

Marchand²

2015

J Mol Biomark Diagn

110

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Type 2 diabetes mellitus (T2DM) is the most common metabolic disorder worldwide. Because of population aging and increasing trends toward obesity and sedentary lifestyles, the number of affected individuals is increasing at worrisome rates. While both environmental and genetic factors are known to contribute to the development of T2DM, continuous research is needed to identify specific biomarkers that could aid both in prevention of the disease and development of newer therapeutic options. Circulating miRNAs are considered as potential biomarkers because they are stable and resistant to degradation by blood RNAses and are modified under different pathophysiological conditions. In this study we carried out a systematic electronic search on PubMed to retrieve all articles that have investigated circulating miRNAs for diagnosing obesity andT2DM in human. We also included lifestyle intervention studies known to be highly effective in delaying onset of diabetes, and studies analyzing the effect of bariatric surgery and anti-diabetic treatment. A total of 26 studies were enrolled in the global meta-analysis. Candidate miRNAs were defined as those reported in at least 2 studies with same direction of differential expression. Ten miRNAs altered in blood of patients suffering fromT2DM were identified (increased: miR-320a, miR-142-3p, miR-222, miR-29a, miR-27a, miR-375; decreased: miR-197, miR-20b, miR-17, miR-652) and 7 miRNAs in blood of obese subjects were identified (increased: miR-142-3p, miR-140-5p, miR-222; decreased:miR-21-5p, miR-221-3p, miR-125-5p, mir-103-5p). Both obese and T2DM patients had elevated concentrations of miR-142-3p and miR-222. MiRNAs target genes were predicted and their cellular functions are discussed in relation with the pathologies. Although a significant number of studies were taken into account in this review, we found a strong discrepancy between miRNA detection and quantification indicating that many of pre-analytical variables have yet to be normalized. Pre-analytical and analytical challenges are also discussed.

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Section: Discussionmentioning

confidence: 99%

Diagnostic Value of Cell-free Circulating Micrornas for Obesity and Type 2 Diabetes: A Meta-analysis

Villard¹,

Marchand²

2015

J Mol Biomark Diagn

110

View full text Add to dashboard Cite

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“…Nevertheless, high-quality serum miRNA extraction is the first step that decides whether serum miRNA is effective for diagnosis, and also directly decides the accuracy of diagnosis results. There are many methods for extraction of serum miRNAs 34, 35, including commercial kits, phenol-chloroform, and Trizol extraction. Most studies state that commercial kits are very effective and outperform the phenol-chloroform method and the Trizol method in terms of simple steps, no addition of reagent or centrifugation, time-saving and labor-saving, higher efficiency and higher yield.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Identification of miRNAs in exosomes derived from serum of colon cancer patients

Zhao

Wang

et al. 2017

J. Cancer

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Objective: To isolate exosomes from the serum of colon cancer patients and identify RNAs in the small vesicles.Methods: ExoQuick-TC™ Exosome Precipitation Solution was used for exosome isolation and the shapes of exosomes were observed under a transmission electron microscope. Mass spectrometry was used to identify the classification of miRNAs encapsulated in exosomes, and the expression levels of miRNA-21,-133a, and -181b in exosomes were detected by RT-PCR.Results: Exosomes isolated from serum of colon cancer patients were circular-or oval-shaped and vary in size with a diameter of 40-100 nm. Mass spectrometry shows that the main RNAs in exosomes are small RNAs; the levels of these small RNAs in exosomes are significantly higher compared with fresh serum. There is still a tiny amount of small RNAs in exosome-free serum, but the amounts are significantly lower than that in exosomes. No more RNAs were detected in the repeated freezing and thawing serum, but there were still some RNAs detectable in the exosomes extracted from these serums. MiRNA-21, -133a and -181b can be detected in exosomes, and the level of miRNA-21 is associated with early diagnosis of colon cancer.Conclusion: This study proves that commercial kits for exosome separation are more convenient and time-saving and that mass spectrometry is capable of identifying the miRNAs in exosomes. Compared with direct extraction of miRNAs from the serum, the method of isolating exosomes from the serum firstly and then extracting miRNAs from the exosomes can enhance the stability and integrity of their inner miRNAs. Also, we demonstrate that the exosomal miRNA-21 expression is associated with the early diagnosis of colon cancer.

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“…The setup of this study is illustrated in Fig 1 and S1 Table. To assess the quantity and the quality of RNA that could be extracted from PBMCs, we stored aliquots containing 10 6 PBMCs for two different time periods (1 day and 83 days) in five different storage media, i.e., (1) at 4˚C in RNAlater (RNL) or at -80˚C in (2) QIAzol Lysis Reagent (QZL, Qiagen), or in the cognate lysis buffers, i.e., (3) easyMAG lysis buffer (EML) or (4) RNeasy Mini Kit lysis buffer (RLT) or (5) RiboPure RNA Purification Kit-blood, lysis buffer (RPL). In addition, three commercial RNA extraction kits were compared, i.e., NucliSENS easyMAG (EM), RNeasy Mini Kit (RE) and RiboPure RNA Purification Kit-blood (RP).…”

Section: General Set Up Of the Comparisonmentioning

confidence: 99%

“…High quality and quantity of extracted RNA is crucial for downstream applications such as reliable gene expression quantification through quantitative PCR [1] and RNAsequencing (RNA-seq) [2,3]. Unfortunately, proper RNA quality controls including RNA integrity are lacking in many studies [4,5,6,7,8].…”

Section: Introductionmentioning

confidence: 99%

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

et al. 2020

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RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (Nucli-SENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4˚C for RNAlater or -80˚C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.PLOS ONE | https://doi.org/10.1371/journal.pone.

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Variability in microRNA recovery from plasma: Comparison of five commercial kits

Cited by 64 publications

References 28 publications

Diagnostic Value of Cell-free Circulating Micrornas for Obesity and Type 2 Diabetes: A Meta-analysis

Diagnostic Value of Cell-free Circulating Micrornas for Obesity and Type 2 Diabetes: A Meta-analysis

Isolation and Identification of miRNAs in exosomes derived from serum of colon cancer patients

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

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