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PloS one2024; 19(5); e0294089; doi: 10.1371/journal.pone.0294089

Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity.

Abstract: Hair follicles provide an easily accessible tissue for interrogating gene expression for multiple purposes in mammals. RNAlater® is a liquid storage solution that stabilises and preserves cellular RNA, eliminating the need to immediately process or freeze tissue specimens. The manufacturer advises storage of samples at 2-8°C overnight before transfer to -20°C. This study aimed to evaluate RNA integrity in hair follicle samples collected from horses, stabilized in RNAlater®, and stored under three short-term storage conditions. Mane hair samples complete with follicles were collected from four horses at a single time point. Approximately 15 hairs were placed in each of three 2 mL tubes containing 0.75ml RNAlater® solution. Test group A was stored at 4°C for 24-h, then decanted and stored at -20°C. Test groups B and C were stored at 4°C and 19°C (room temperature) respectively for 7 days, then decanted and stored at -20°C. RNA was isolated from all samples and RNA quantity and quality were measured. One-way ANOVA revealed no difference in RNA concentration (A:516 +/-125 ng/ml, B:273+/-93 ng/ml, C:476+/-176 ng/ml;P = 0.2) or quality (A:9.5 +/-0.19, B:9.8+/-0.09, C:9.2+/-0.35 RIN; P = 0.46) between the test groups. There were no group differences in mean Cycle Threshold values from qPCR validation assays confirming high-quality template cDNA. The results suggest that storage of hair follicles for one week in RNAlater® at cool or room temperature conditions will not compromise RNA integrity and will permit extended transport times from remote sampling locations without the need for freezing.
Copyright: © 2024 Harkin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Publication Date: 2024-05-31 PubMed ID: 38820307PubMed Central: PMC11142484DOI: 10.1371/journal.pone.0294089Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigated how the storage conditions of horse hair follicles in RNAlater® affect RNA integrity over short periods.
  • The research showed that RNA quality and quantity remain stable even after one week at cool or room temperature, supporting easier transport and storage without immediate freezing.

Introduction

  • Hair follicles are a convenient biological sample to analyze gene expression because they are easy to collect from mammals.
  • Analyzing RNA from hair follicles helps in various biological and medical studies.
  • RNA is sensitive and can degrade quickly unless preserved properly after collection.
  • RNAlater® is a commercial liquid solution designed to stabilize and protect RNA immediately after tissue collection, simplifying handling and storage.
  • The manufacturer recommends storing samples initially at 2-8°C overnight before transferring them to -20°C for longer term storage.
  • The goal of this study was to test if RNA integrity is maintained under different short-term storage temperatures using RNAlater®, potentially extending flexibility in sample handling.

Methods

  • Samples were collected from four horses by plucking mane hair follicles; about 15 hairs with follicles were placed per tube.
  • Each sample tube contained 0.75 mL of RNAlater® solution to stabilize RNA.
  • Three test groups were designed based on storage conditions before freezing:
    • Test Group A: Stored at 4°C for 24 hours, then solution decanted and samples frozen at -20°C.
    • Test Group B: Stored at 4°C for 7 days, then decanted and frozen at -20°C.
    • Test Group C: Stored at 19°C (room temperature) for 7 days, then decanted and frozen at -20°C.
  • RNA was extracted from all samples after the storage period.
  • RNA quantity (concentration) and quality were measured using standard laboratory methods.
  • Quantitative PCR (qPCR) was used to validate the quality of complementary DNA (cDNA) synthesized from RNA templates by measuring Cycle Threshold (Ct) values.
  • Statistical analysis: One-way ANOVA was used to compare RNA quality and quantity across groups.

Results

  • No significant differences in RNA concentration between groups:
    • Group A: 516 ± 125 ng/mL
    • Group B: 273 ± 93 ng/mL
    • Group C: 476 ± 176 ng/mL
    • p = 0.2 (not statistically significant)
  • No significant differences in RNA integrity number (RIN) values, indicating RNA quality:
    • Group A: 9.5 ± 0.19
    • Group B: 9.8 ± 0.09
    • Group C: 9.2 ± 0.35
    • p = 0.46 (not statistically significant)
  • qPCR Cycle Threshold (Ct) values showed no group differences, confirming high-quality cDNA templates suitable for gene expression analysis.

Conclusions and Implications

  • Storing hair follicles in RNAlater® for up to one week at either 4°C or room temperature does not significantly degrade RNA integrity or reduce RNA quantity.
  • This finding increases the practicality of collecting hair follicle samples remotely where immediate freezing is not possible.
  • Samples can be safely transported for up to seven days without freezing, which can reduce costs and logistical challenges of shipping biological material.
  • Overall, RNAlater® provides effective preservation of RNA in hair follicle samples under varied short-term storage conditions.

Cite This Article

APA
Harkin EE, Browne JA, Murphy BA. (2024). Evaluation of short-term hair follicle storage conditions for maintenance of RNA integrity. PLoS One, 19(5), e0294089. https://doi.org/10.1371/journal.pone.0294089

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 19
Issue: 5
Pages: e0294089
PII: e0294089

Researcher Affiliations

Harkin, Eilís E
  • School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland.
Browne, John A
  • School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland.
Murphy, Barbara A
  • School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland.

MeSH Terms

  • Hair Follicle / metabolism
  • Animals
  • Horses
  • RNA / genetics
  • RNA / analysis
  • RNA Stability
  • Specimen Handling / methods
  • Time Factors
  • Temperature
  • Cryopreservation / methods

Conflict of Interest Statement

The authors have declared that no competing interests exist.

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Citations

This article has been cited 1 times.
  1. Greening L, Harkin E, Kyriazopoulou P, Heppelthwaite Z, Aragona F, Browne JA, Hemmings A, Williams JM, Murphy BA. Influence of lighting on sleep behaviour, circadian rhythm and spontaneous blink rate in stabled riding school horses (Equus caballus). PLoS One 2025;20(6):e0326567.
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