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Published (Version of record)CC BY V4.0, Open Access
Published (Version of record)CC BY V4.0, Open
Journal article
Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system
Diagnostic Pathology, Vol.15(1), pp.1-7
15/05/2020
PMCID: PMC7229590
PMID: 32414387
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Source: InCites
Abstract
Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System's ability to reproducibly quantify the expression of 730 immune genes from skin biopsies.
Healthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at - 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at - 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at - 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes.
Except for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols.
A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.
Details
- Title
- Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system
- Creators
- Jelena Vider - Griffith UniversityAndrew Croaker - Southern Cross UniversityAmanda J Cox - Griffith UniversityEmma Raymond - Wesley Medical ResearchRebecca Rogers - Wesley Medical ResearchStuart Adamson - Mid-West Aero Medical, Geraldton, Perth AustraliaMichael Doyle - Wesley Medical ResearchBlake O’Brien - Sullivan &Nicolaides Pathology, Bowen Hills, QLD AustraliaAllan W Cripps - Griffith UniversityNicholas P West - Griffith University
- Publication Details
- Diagnostic Pathology, Vol.15(1), pp.1-7
- Publisher
- BioMed Central Ltd.; London
- Grant note
- This work received funding support from the Skin Cancer College Australasia; the non-profit peak body supporting education, research, advocacy and standards for primary care skin cancer professionals in Australia and New Zealand.
- Identifiers
- 991012925024202368
- Copyright
- © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
- Academic Unit
- Faculty of Health; School of Health and Human Sciences
- Language
- English
- Resource Type
- Journal article