Identification of Endogenous Controls for Analyzing Serum Exosomal miRNA in Patients with Hepatitis B or Hepatocellular Carcinoma

Li, Yang; Zhang, Liqun; Liu, Fei; Xiang, Guiming; Jiang, Dongneng; Pu, Ximing

doi:10.1155/2015/893594

Cited by 86 publications

(62 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…△CT was used to show the differences between target miRNAs and internal reference (small nucleolar RNA, RNU6). The amounts of target miRNAs in EVs were expressed as CT values [29]. …”

Section: Methodsmentioning

confidence: 99%

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line

et al. 2017

View full text Add to dashboard Cite

Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.

show abstract

“…△CT was used to show the differences between target miRNAs and internal reference (small nucleolar RNA, RNU6). The amounts of target miRNAs in EVs were expressed as CT values [29]. …”

Section: Methodsmentioning

confidence: 99%

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line

et al. 2017

View full text Add to dashboard Cite

show abstract

“…To find out the control gene for exosomal miRNA normalization, Le et al evaluated the expression stability of 11 reference genes in circulating exosomes, and found that the combination of miR-221, miR-191, let-7a, miR-181a, and miR-26a can be an optimal gene reference set for normalizing the expression of liver-specific miRNAs. This combination enhanced the robustness of the relative quantification analyses (82). …”

Section: Relative Data Normalization To Endogenous Reference Genesmentioning

confidence: 98%

Data Normalization Strategies for MicroRNA Quantification

et al. 2015

View full text Add to dashboard Cite

Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure miRNA expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains to be problematic, and can have serious impact on the actual available transcript levels and consequently, on the biological interpretation of data. In this review article we intend to discuss the reliability of use of small RNAs commonly reported in the literature as miRNAs expression normalizers and to compare different normalization strategies used, setting the base for establishing a global standard of miRNAs expression data normalization.

show abstract

“…geNorm and NormFinder were also employed to investigate on a set of reference genes (5S, U6, hsa-let-7a, hsa-miR-103, hsa-miR-16, hsa-miR-181a, hsa-miR-181c, hsa-miR-191, hsa-miR-22, hsa-miR-221, and hsa-miR-26a) to analyze exosome-associated miRNAs in serum of patients with hepatitis B or hepatocellular carcinoma. The two algorithms have identified the combination of hsa-miR-221, hsa-miR-191, hsa-let-7a, hsa-miR-181a, hsa-miR-181c, and hsa-miR-26a as the optimal reference genes to normalize liver-derived miRNAs [99]. In a recent study on fracture riskassociated miRNA, we used miR-425-5p and miR-484 as reference genes according to a priori analysis [21].…”

Section: Normalization On Endogenous Reference Genesmentioning

confidence: 99%