FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / PLoS One / Evaluation of short-term hair follicle storage conditions fo...
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.
Get Full Text
Publication Date: 2024-05-31 PubMed ID: 38820307PubMed Central: PMC11142484DOI: 10.1371/journal.pone.0294089Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

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.

References

This article includes 36 references
  1. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M. The RIN: an RNA integrity number for assigning integrity values to RNA measurements.. BMC Mol Biol 2006;7(1):3.
    doi: 10.1186/1471-2199-7-3pmc: PMC1413964pubmed: 16448564google scholar: lookup
  2. Ho Holland NT, Smith MT, Eskenazi B, Bastaki M. Biological sample collection and processing for molecular epidemiological studies.. Mutat Res 2003. Jun;543(3):217–34.
    doi: 10.1016/s1383-5742(02)00090-xpubmed: 12787814google scholar: lookup
  3. Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S. Measurable impact of RNA quality on gene expression results from quantitative PCR.. Nucleic Acids Res 2011. May;39(9):e63.
    doi: 10.1093/nar/gkr065pmc: PMC3089491pubmed: 21317187google scholar: lookup
  4. Lightfoot S, Salowsky R, Buhlmann C. RNA integrity number: towards standardization of RNA quality assessment for better reproducibility and reliability of gene expression experiments.. Breast Cancer Res 2005;7(S2).
  5. LifeTechnologies. RNAlater® Tissue Collection: RNA StabilizationSolution. 2011.
  6. Sigma-Aldrich. Technical Bulletin. 2016.
  7. Drakulovski P, Locatelli S, Butel C, Pion S, Krasteva D, Mougdi-Pole E. Use of RNAlater® as a preservation method for parasitic coprology studies in wild-living chimpanzees.. Exp Parasitol 2013;135(2):257–61.
    doi: 10.1016/j.exppara.2013.07.002pmc: PMC3907073pubmed: 23850999google scholar: lookup
  8. Nicoletti A, Pregel P, Starvaggi Cucuzza L, Cannizzo FT, Sereno A, Scaglione FE. Coping with tissue sampling in suboptimal conditions: Comparison of different tissue preservation methods for histological and molecular analysis.. Animals 2021;11(3):649.
    doi: 10.3390/ani11030649pmc: PMC8001879pubmed: 33804460google scholar: lookup
  9. Prescott MJ, Lidster K. Improving quality of science through better animal welfare: the NC3Rs strategy.. Lab Animal 2017;46(4):152–6.
    doi: 10.1038/laban.1217pubmed: 28328893google scholar: lookup
  10. Kudlova N, Slavik H, Duskova P, Furst T, Srovnal J, Bartek J. An efficient, non-invasive approach for in-vivo sampling of hair follicles: design and applications in monitoring DNA damage and aging.. Aging 2021;13(23):25004–24.
    doi: 10.18632/aging.203744pmc: PMC8714131pubmed: 34874896google scholar: lookup
  11. Green MR, Clay CS, Gibson WT, Hughes TC, Smith CG, Westgate GE. Rapid isolation in large numbers of intact, viable, individual hair follicles from skin: biochemical and ultrastructural characterization.. J Invest Dermatol 1986. Dec;87(6):768–70.
    doi: 10.1111/1523-1747.ep12457348pubmed: 3782859google scholar: lookup
  12. Karstensen JG, Vilmann P. Historical perspective on needle development: From the past to the future.. Best Pract Res Clin Gastroenterol 2022. Sep-Dec;60–61:101814.
    doi: 10.1016/j.bpg.2022.101814pubmed: 36577533google scholar: lookup
  13. Zhang J, Wallace SJ, Shiu MY, Smith I, Rhind SG, Langlois VS. Human hair follicle transcriptome profiling: a minimally invasive tool to assess molecular adaptations upon low‐volume, high‐intensity interval training.. Physiol Rep 2017;5(23):e13534.
    doi: 10.14814/phy2.13534pmc: PMC5727284pubmed: 29212859google scholar: lookup
  14. Watts LM, Browne JA, Murphy BA. Investigation of a non-invasive method of assessing the equine circadian clock using hair follicle cells.. J Circadian Rhythms 2012;10(0):7.
    doi: 10.1186/1740-3391-10-7pmc: PMC3514281pubmed: 23039139google scholar: lookup
  15. Henry P, Henry A, Russello MA. A noninvasive hair sampling technique to obtain high quality DNA from elusive small mammals.. J Vis Exp 2011;13;(49):2791.
    doi: 10.3791/2791pmc: PMC3197329pubmed: 21445038google scholar: lookup
  16. Collery A, Browne JA, O’Brien C, Sheridan JT, Murphy BA. Optimised stable lighting strengthens circadian clock gene rhythmicity in equine hair follicles. Animals. 2023;13(14):2335. doi: 10.3390/ani13142335
    doi: 10.3390/ani13142335pmc: PMC10376498pubmed: 37508112google scholar: lookup
  17. Bradley BJ, Pastorini J, Mundy NI. Successful retrieval of mRNA from hair follicles stored at room temperature: implications for studying gene expression in wild mammals. Mol Ecol Notes. 2005;5(4):961–4.
  18. Kim SJ, Dix DJ, Thompson KE, Murrell RN, Schmid JE, Gallagher JE, et al. Gene expression in head hair follicles plucked from men and women. Ann Clin Lab Sci. 2006. Spring;36(2):115–26.
    pubmed: 16682506
  19. Lorenzetti WR, Ibelli AMG, Peixoto JDO, Mores MAZ, Savoldi IR, Carmo KBD, et al. Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs. PLoS ONE. 2018;13(9):e0204348. doi: 10.1371/journal.pone.0204348
    doi: 10.1371/journal.pone.0204348pmc: PMC6147718pubmed: 30235332google scholar: lookup
  20. Neary MT, Neary JM, Lund GK, Holt TN, Mohun TJ, Breckenridge RA, et al. Technical Note: A comparison of DNA collection methods in cattle and Yaks. J Animal Sci 2014;92(9):3811–3815. doi: 10.2527/jas.2013-7445
    doi: 10.2527/jas.2013-7445pubmed: 25085402google scholar: lookup
  21. Wang GY, Wang J, Mancianti M-L, Epstein EH. Basal Cell Carcinomas Arise from Hair Follicle Stem Cells in Ptch1+/− Mice. Cancer Cell. 2011;19(1):114–24. doi: 10.1016/j.ccr.2010.11.007
    doi: 10.1016/j.ccr.2010.11.007pmc: PMC3061401pubmed: 21215705google scholar: lookup
  22. Zhang J, Carnduff L, Norman G, Josey T, Wang Y, Sawyer TW, et al. Transcriptional profiling in rat hair follicles following simulated blast insult: A new diagnostic tool for traumatic brain injury. PLoS ONE. 2014;9(8):e104518. doi: 10.1371/journal.pone.0104518
    doi: 10.1371/journal.pone.0104518pmc: PMC4138085pubmed: 25136963google scholar: lookup
  23. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006;27(2–3):126–39. doi: 10.1016/j.mam.2005.12.003
    doi: 10.1016/j.mam.2005.12.003pubmed: 16469371google scholar: lookup
  24. Liu Q, Tang Y, Huang Y, Wang J, Yang K, Zhang Y, et al. Insights into male androgenetic alopecia using comparative transcriptome profiling: hypoxia-inducible factor-1 and Wnt/β-catenin signalling pathways. Br J Dermatol. 2022;187(6):936–47.
    pmc: PMC10087000pubmed: 35862273
  25. Gu X, Han Y, Shao Y, Ma W, Shao Z, Wan G, et al. Gene expression changes reveal the impact of the space environment on the skin of international space station astronauts. Clin Exp Dermatol. 2023;48(10):1128–1137. doi: 10.1093/ced/llad178
    doi: 10.1093/ced/llad178pubmed: 37171787google scholar: lookup
  26. Naboulsi R, Cieślak J, Headon D, Jouni A, Negro JJ, Andersson G, et al. The enrichment of specific hair follicle-associated cell populations in plucked hairs offers an opportunity to study gene expression underlying hair traits. Int J Mol Sci. 2022;24(1):561. doi: 10.3390/ijms24010561
    doi: 10.3390/ijms24010561pmc: PMC9820680pubmed: 36614000google scholar: lookup
  27. O’Brien C, Darcy-Dunne MR, Murphy BA. The effects of extended photoperiod and warmth on hair growth in ponies and horses at different times of year. PLoS ONE. 2020;15(1):e0227115. doi: 10.1371/journal.pone.0227115
    doi: 10.1371/journal.pone.0227115pmc: PMC6959597pubmed: 31935219google scholar: lookup
  28. Rieu I, Powers SJ. Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics. The Plant Cell. 2009;21(4) 1031–33. doi: 10.1105/tpc.109.066001
    doi: 10.1105/tpc.109.066001pmc: PMC2685626pubmed: 19395682google scholar: lookup
  29. Akashi M, Soma H, Yamamoto T, Tsugitomi A, Yamashita S, Yamamoto T, et al. Noninvasive method for assessing the human circadian clock using hair follicle cells. Proc Natl Acad Sci. 2010;107(35):15643–8. doi: 10.1073/pnas.1003878107
    doi: 10.1073/pnas.1003878107pmc: PMC2932591pubmed: 20798039google scholar: lookup
  30. Liu L-P, Li M-H, Zheng Y-W. Hair follicles as a critical model for monitoring the circadian clock. Int J Mol Sci [Internet]. 2023. Jan 26;24(3):2407. doi: 10.3390/ijms24032407
    doi: 10.3390/ijms24032407pmc: PMC9916850pubmed: 36768730google scholar: lookup
  31. Al-Nuaimi Y, Hardman JA, Bíró T, Haslam IS, Philpott MP, Tóth BI, et al. A meeting of two chronobiological systems: Circadian proteins Period1 and BMAL1 modulate the human hair cycle clock. J Investig Dermatol. 2014;134(3):610–9. doi: 10.1038/jid.2013.366
    doi: 10.1038/jid.2013.366pubmed: 24005054google scholar: lookup
  32. Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature. 2002;418(6901):935–41. doi: 10.1038/nature00965
    doi: 10.1038/nature00965pubmed: 12198538google scholar: lookup
  33. Husse J, Eichele G, Oster H. Synchronization of the mammalian circadian timing system: Light can control peripheral clocks independently of the SCN clock. BioEssays. 2015;37(10):1119–28.
    pmc: PMC5054915pubmed: 26252253
  34. Richards J, Gumz ML. Advances in understanding the peripheral circadian clocks. FASEB J. 2012;26(9):3602–13. doi: 10.1096/fj.12-203554
    doi: 10.1096/fj.12-203554pmc: PMC3425819pubmed: 22661008google scholar: lookup
  35. Baron KG, Reid KJ. Circadian misalignment and health. Int Rev Psychiatry. 2014;26(2):139–54. doi: 10.3109/09540261.2014.911149
    doi: 10.3109/09540261.2014.911149pmc: PMC4677771pubmed: 24892891google scholar: lookup
  36. Wolfensohn S, Shotton J, Bowley H, Davies S, Thompson S, Justice W, et al. Assessment of welfare in zoo animals: Towards optimum quality of life. Animals. 2018;8(7):110. doi: 10.3390/ani8070110
    doi: 10.3390/ani8070110pmc: PMC6071229pubmed: 29973560google scholar: lookup

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.
    doi: 10.1371/journal.pone.0326567pubmed: 40577374google scholar: lookup
Subscribe to our newsletter
Join 99,030 horse owners like you who receive our email updates on horse health and nutrition.