Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank

Ortega-Pinazo, Jesús; Díaz, Tatiana; Martínez, Beatriz; Jiménez, Álvaro; Pinto-Medel, María Jesús; Ferro, P.

doi:10.1007/s10561-019-09764-9

Cited by 9 publications

(7 citation statements)

References 54 publications

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“…Our assay was optimized for analysis using cryopreserved PBMCs since these are common sample sources for human immunoassays ( 24 ), as blood collection is less-invasive ( 25 ) and PBMC isolation is relatively technically straightforward ( 26 ) and cost-efficient ( 27 ). Additionally, cryopreserved PBMCs can be shipped globally ( 28 ) for batched testing ( 29 ) or long-term storage in biobanks ( 30 ). It is reasonable to expect an even higher diagnostic sensitivity than reported herein could be achieved using our cost-optimized RT-qPCR protocol with fresh PBMCs, as cryopreservation can profoundly influence surface marker and antigen-specific T cell responses ( 5 , 24 ).…”

Section: Discussionmentioning

confidence: 99%

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

et al. 2022

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Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

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Section: Discussionmentioning

confidence: 99%

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

et al. 2022

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“…Cell cultures analyzes were performed in triplicates. The procedure was carried out as previously described by Ortega-Pinazo et al [26].…”

Section: Cell Culturesmentioning

confidence: 99%

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices

Ortega-Pinazo,

Pacheco-Rodríguez,

Serrano-Castro

et al. 2023

Mol Biol Rep

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Nucleic acids, RNA among them, are widely used in biomedicine and Biotechnology.Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. QIAcube™ and semi-automated extraction methods were perceived as the best options because of their lower variability, good functionality, and lower cost (P<0.001).These data contribute to facilitating researchers or research service platforms (Biobanks) in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

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“…The study was carried out in accordance with The Code of Ethics of the WMA (Declaration of Helsinki). The procedure was carried out as previously described by Ortega-Pinazo et al [26].…”

Section: Subjectsmentioning

confidence: 99%

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

Ortega-Pinazo

Serrano-Castro

Martínez

et al. 2022

Preprint

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Nucleic acids, RNA among them, are widely used in biomedicine and biotechnology. Because of their susceptibility to degradation by RNases, the handling and extraction process of RNA from cells and tissues require specialized personnel and standardized methods to guarantee high purity and integrity. Due to the diversity of techniques found in the market, a comparative study between different RNA extraction methods is useful to facilitate the best choice for the researcher or in research service platforms such as biobanks to see the traceability of the samples. In this study, we have compared seven different RNA extraction methods: manual (TRIzol™), semiautomated (QIAGEN™, Bio-Rad, Monarch®, and Canvax™), and fully automated (QIAcube™ and Maxwell®) processes, from two biological matrices: human Jurkat T cells and peripheral blood mononuclear cells (PBMC). Results showed marked differences in the RNA quality and functionality according to the method employed for RNA extraction and the matrix used. These data contribute to facilitating researchers or research service platforms in decision-making practices and emphasize the relevance of the selection of the RNA extraction method in each experimental procedure or traceability study to guarantee both quality standards and its reproducibility.

show abstract

Quality assessment on the long-term cryopreservation and nucleic acids extraction processes implemented in the andalusian public biobank

Cited by 9 publications

References 54 publications

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices

Comparing RNA extraction methods to face the variations in RNA quality using two human biological matrices.

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