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Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation.

ABSTRACT: microRNAs are promising biomarkers in biological fluids in several diseases. Different plasma RNA isolation protocols and carriers are available, but their efficiencies have been scarcely compared. Plasma microRNAs were isolated using a phenol and column-based procedure and a column-based procedure, in the presence or absence of two RNA carriers (yeast RNA and MS2 RNA). We evaluated the presence of PCR inhibitors and the relative abundance of certain microRNAs by qRT-PCR. Furthermore, we analyzed the association between different isolation protocols, the relative abundance of the miRNAs in the sample, the GC content and the free energy of microRNAs. In all microRNAs analyzed, the addition of yeast RNA as a carrier in the different isolation protocols used gave lower raw Cq values, indicating higher microRNA recovery. Moreover, this increase in microRNAs recovery was dependent on their own relative abundance in the sample, their GC content and the free-energy of their own most stable secondary structure. Furthermore, the normalization of microRNA levels by an endogenous microRNA is more reliable than the normalization by plasma volume, as it reduced the difference in microRNA fold abundance between the different isolation protocols evaluated. Our thorough study indicates that a standardization of pre- and analytical conditions is necessary to obtain reproducible inter-laboratory results in plasma microRNA studies.

SUBMITTER: Ramon-Nunez LA

PROVIDER: S-EPMC5659774 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The successful translation of mesenchymal stem cells (MSCs) from bench to bedside is predicated upon their regenerative capabilities and immunomodulatory potential. Many challenges still exist in making MSCs a viable and cost-effective therapeutic option, due in part to the challenges of sourcing MSCs from adult tissues and inconsistencies in the characterization of MSCs. In many cases, adult MSC collection is an invasive procedure, and ethical concerns and age-related heterogeneity further complicate obtaining adult tissue derived MSCs at the scales needed for clinical applications. Alternative adult sources, such as post-partum associated tissues, offer distinct advantages to overcome these challenges. However, successful therapeutic applications rely on the efficient ex-vivo expansion of the stem cells while avoiding any culture-related phenotypic alterations, which requires optimized and standardized isolation, culture, and cell preservation methods. In this review, we have compared the isolation and culture methods for MSCs originating from the human amniotic membrane (hAMSCs) of the placenta to identify the elements that support the extended subculture potential of hAMSCs without compromising their immune-privileged, pluripotent regenerative potential.Abbreviations: AM: Human amniotic membrane; ASCs: Adipose tissue-derived stem cells; BM-MSCs: Bone marrow-mesenchymal stem cells; DMEM: Dulbecco's modified eagle medium; DT: Doubling time; EMEM: Eagle's modified essential medium; ESCM: Embryonic stem cell markers; ESCs: Embryonic stem cells; hAECs: Human amniotic epithelial cells; hAMSCs: Human amniotic mesenchymal stem cells; HLA: Human leukocyte antigen; HM: Hematopoietic markers; IM: Immunogenicity markers; MHC: Major histocompatibility complex; MSCs: Mesenchymal stem cells; MCSM: Mesenchymal cell surface markers; Nanog: NANOG homeobox; Oct: Octamer binding transcription factor 4; P: Passage; PM: Pluripotency markers; STRO-1: Stromal precursor antigen-1; SCP: Subculture potential; Sox-2: Sry-related HMG box gene 2; SSEA-4: Stage-specific embryonic antigen; TRA: Tumor rejection antigen.

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Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation.

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