Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations

Mirna Andelic; Margherita Marchi; Stefania Marcuzzo; Raffaella Lombardi; Catharina G. Faber; Giuseppe Lauria; Erika Salvi

doi:10.1016/j.omtm.2023.101116

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations

Mirna Andelic, Margherita Marchi, Stefania Marcuzzo, Raffaella Lombardi, Catharina G. Faber, Giuseppe Lauria, Erika Salvi^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

Original language	English
Article number	101116
Number of pages	12
Journal	Molecular Therapy - Methods and Clinical Development
Volume	31
Issue number	1
Early online date	28 Sept 2023
DOIs	https://doi.org/10.1016/j.omtm.2023.101116
Publication status	Published - 14 Dec 2023

Keywords

archival specimens
epidermal miRNA reference
fixed skin biopsy
microfluidic card
miRNA profiling
post-analytical settings
RT-qPCR post-analysis settings

Access to Document

10.1016/j.omtm.2023.101116Licence: CC BY

Cite this

@article{a323f778d3d943d8bb76e7125201d261,

title = "Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations",

abstract = "Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.",

keywords = "archival specimens, epidermal miRNA reference, fixed skin biopsy, microfluidic card, miRNA profiling, post-analytical settings, RT-qPCR post-analysis settings",

author = "Mirna Andelic and Margherita Marchi and Stefania Marcuzzo and Raffaella Lombardi and Faber, {Catharina G.} and Giuseppe Lauria and Erika Salvi",

note = "Funding Information: This work was supported with PAIN-net Project: Molecule-to-Man Pain Network, EU Research Framework Programme H2020 /Marie Sklodowska-Curie Actions, grant agreement number 721841 and Italian Ministry of Health (RRC), Italy. Funding Information: We thank Dr. Rosina Paterra from the Molecular Neuro-Oncology Unit (Fondazione IRCCS Istituto Neurologico Carlo Besta) for advice and technical support with RNA isolation. We thank the technical support of Thermo Fisher Scientific for troubleshooting. The authors also thank Rosalind Hendricks, Assistant Biomedical Librarian of the “Fondazione IRCCS Istituto Neurologico Carlo Besta,” for English language revision of our article. This work was supported with PAIN-net Project: Molecule-to-Man Pain Network, EU Research Framework Programme H2020/Marie Sklodowska-Curie Actions, grant agreement number 721841 and Italian Ministry of Health (RRC), Italy. M.A. and E.S. conceptualized the study. M.A. M.M. and S.M. designed and M.A. performed the experiments. R.L. processed all specimens. E.S. performed the data analysis. G.L. and C.F. performed supervision and funding acquisition. All the authors wrote and edited the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2023 The Author(s)",

year = "2023",

month = dec,

day = "14",

doi = "10.1016/j.omtm.2023.101116",

language = "English",

volume = "31",

journal = "Molecular Therapy - Methods and Clinical Development",

issn = "2329-0501",

publisher = "Nature Publishing Group",

number = "1",

}

TY - JOUR

T1 - Archival skin biopsy specimens as a tool for miRNA-based diagnosis

T2 - Technical and post-analytical considerations

AU - Andelic, Mirna

AU - Marchi, Margherita

AU - Marcuzzo, Stefania

AU - Lombardi, Raffaella

AU - Faber, Catharina G.

AU - Lauria, Giuseppe

AU - Salvi, Erika

N1 - Funding Information: This work was supported with PAIN-net Project: Molecule-to-Man Pain Network, EU Research Framework Programme H2020 /Marie Sklodowska-Curie Actions, grant agreement number 721841 and Italian Ministry of Health (RRC), Italy. Funding Information: We thank Dr. Rosina Paterra from the Molecular Neuro-Oncology Unit (Fondazione IRCCS Istituto Neurologico Carlo Besta) for advice and technical support with RNA isolation. We thank the technical support of Thermo Fisher Scientific for troubleshooting. The authors also thank Rosalind Hendricks, Assistant Biomedical Librarian of the “Fondazione IRCCS Istituto Neurologico Carlo Besta,” for English language revision of our article. This work was supported with PAIN-net Project: Molecule-to-Man Pain Network, EU Research Framework Programme H2020/Marie Sklodowska-Curie Actions, grant agreement number 721841 and Italian Ministry of Health (RRC), Italy. M.A. and E.S. conceptualized the study. M.A. M.M. and S.M. designed and M.A. performed the experiments. R.L. processed all specimens. E.S. performed the data analysis. G.L. and C.F. performed supervision and funding acquisition. All the authors wrote and edited the manuscript. The authors declare no competing interests. Publisher Copyright: © 2023 The Author(s)

PY - 2023/12/14

Y1 - 2023/12/14

N2 - Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

AB - Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

KW - archival specimens

KW - epidermal miRNA reference

KW - fixed skin biopsy

KW - microfluidic card

KW - miRNA profiling

KW - post-analytical settings

KW - RT-qPCR post-analysis settings

U2 - 10.1016/j.omtm.2023.101116

DO - 10.1016/j.omtm.2023.101116

M3 - Article

SN - 2329-0501

VL - 31

JO - Molecular Therapy - Methods and Clinical Development

JF - Molecular Therapy - Methods and Clinical Development

IS - 1

M1 - 101116

ER -