Optimizing Tear Collection in Mice for mRNA and Protein Analysis
Summary
The identification of RNA and protein biomarkers from tears in mouse models holds great promise for early diagnostics in various diseases. This manuscript provides a comprehensive protocol for optimizing the efficacy and efficiency of mRNA and protein isolation from mouse tears.
Abstract
The tear film is a highly dynamic biofluid capable of reflecting pathology-associated molecular changes, not only in the ocular surface but also in other tissues and organs. Molecular analysis of this biofluid offers a non-invasive way to diagnose or monitor diseases, assess medical treatment efficacy, and identify possible biomarkers. Due to the limited sample volume, collecting tear samples requires specific skills and appropriate tools to ensure high quality and maximum efficiency. Various tear sampling methodologies have been described in human studies. In this article, a comprehensive description of an optimized protocol is presented, specifically tailored for extracting tear-related protein information from experimental animal models, especially mice. This method includes the pharmacological stimulation of tear production in 2-month-old mice, followed by sample collection using Schirmer strips and the evaluation of the efficacy and efficiency of the protocol through standard procedures, SDS-PAGE, qPCR, and digital PCR (dPCR). This protocol can be easily adapted for the investigation of the tear protein signature in a variety of experimental paradigms. By establishing an affordable, standardized, and optimized tear sampling protocol for animal models, the aim was to bridge the gap between human and animal research, facilitating translational studies and accelerating advancements in the field of ocular and systemic disease research.
Introduction
Tears are considered a plasma ultrafiltrate and have also been described as an intermediate fluid between plasma serum and cerebrospinal fluid due to a significant overlap in the biomolecules they share1. It has been reported that human tears contain proteins, tear lipids, metabolites, and electrolytes2. Recently, other biomolecules such as mRNAs, miRNAs, and extracellular vesicles have also been identified3,4,5,6,7.
In humans, basal tears are located in the tear film, which consists of three layers: the outer lipid layer, which maintains the tear surface smooth to allow us to see through it and prevent tear evaporation; the middle aqueous layer, which keeps the eye hydrated, repels bacteria, protects the cornea, and constitutes 90% of the tear film; and finally, the mucin layer, a family of high molecular weight proteins that are in contact with the cornea and allow the tear to adhere to the eye8. Tear distribution across the ocular surface begins with secretion from the lacrimal gland. This fluid is then guided through tear ducts to pass over the eye's surface and flow into drainage channels. Each blink allows tears to disperse evenly over the entire eye, keeping it moist9.
The tear film is a highly dynamic biofluid capable of reflecting molecular changes that occur not only on the ocular surface but also in other tissues and organs. The analysis of differential expression in this biofluid represents a promising approach for the discovery of biomarkers in human diseases10,11. The utilization of tear film as a source of biomarkers for early diagnosis in various pathologies has been greatly facilitated by the presence of non-invasive collection methods. The most common method for collecting tears in human and veterinary clinics involves membrane-based support (Schirmer's strip), which operates on the principle of capillary action, allowing the water in tears to travel along the length of a paper test strip or capillary tubes placed in the subject's lower conjunctival sac12,13,14. Despite the inherent limitation of obtaining a small sample volume through this method, the biochemical analysis of tear composition using various sensitive techniques has facilitated the identification of potential biomarker molecules11,15. Protocols for optimizing and evaluating the elution of tear proteins from Schirmer strips and capillaries in human patients are well documented16,17. However, complete descriptions of optimized protocols specifically designed to extract molecular information related to tears from experimental animal models are available but scarce. Existing methods, such as tear induction through direct stimulation of the lacrimal gland18, while allowing for the collection of larger volumes, are invasive and can cause discomfort to the animals. Non-invasive methods, such as collecting tears from the ocular surface, have been described as a way to isolate DNA and miRNAs19,21.
This protocol aims to establish a cost-effective and optimized method for collecting and processing tears in mice. The method prioritizes non-invasiveness while obtaining sufficient tear volumes suitable for molecular analysis through techniques like SDS-PAGE, qPCR, and dPCR. The extracted protein and mRNA content information can then be utilized to identify potential biomarkers in existing experimental models of disease.
Protocol
Representative Results
Discussion
Tear fluid is easily accessible, and the determination of biomarkers in tears can be employed as a successful complementary technique for the early diagnosis of various human diseases27. While the biochemical analysis of tear composition in experimental animal models complements this approach and promises significant progress in understanding the molecular basis of diseases, there is a scarcity of available data and protocols, which led us to develop one. The method described in this report is tec…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by VELUX STIFTUNG [project 1852] to M.L. and postgraduate fellowship grants from CONAHCYT to M.B. (836810), E.J.M.C. (802436) and A.M.F (CVU 1317418). Sincere gratitude to all the members of the laboratory, Centro de Investigación sobre el Envejecimiento, and Departamento de Farmacobiología (Cinvestav) for their contributions to the stimulating discussions.
Materials
| 2-mercaptoethanol | Gibco | 1985023 | |
| 2x Laemmli buffer | Bio-Rad | 16-0737 | |
| Acetic Acid | Quimica Meyer | 64-19-7 | |
| Acrylamide | Sigma-Aldrich | A4058 | |
| Bradford Reagent | Sigma-Aldrich | B6916 | |
| Chloroform | Sigma-Aldrich | 1003045143 | |
| Coomassie Blue R 250 | US Biological | 6104-59-2 | |
| Ethanol | Quimica Rique | 64-17-5 | |
| GeneRuler 1kb plus DNA Ladder | Thermofisher scientific | SM1331 | |
| Glycerol | US Biological | G8145 | |
| Glycine | SANTA CRUZ | SC- 29096 | |
| Glycogen | Roche | 10901393001 | |
| HCl | Quimica Rique | 7647-01-0 | |
| Isopropyl alcohol | Quimica Rique | 67-63-0 | |
| Methanol | Quimica Meyer | 67-56-1 | |
| Micro tubes 1.5 ml | Axygen | MCT-150-C | |
| Micro tubes 600 µl | Axygen | MCT-060-C | |
| NaCl | Sigma-Aldrich | S3014 | |
| PCR tubes & strips | Novasbio | PCR 0104 | |
| Pilocarpine | Sigma-Aldrich | P6503-10g | |
| Protease inhibitor | Roche | 11873580001 | |
| QIAcuity EvaGreen PCR Kit (5mL) | Qiagen | 250112 | |
| QIAcuity Nanoplate 26k 24-well (10) | Qiagen | 250001 | |
| Real qPlus 2x Master Mix Green | Ampliqon | A323402 | |
| RevertAid First Strad cDNA Synthesis Kit | Thermofisher scientific | K1622 | |
| Schirmer's test strips | Laboratorio Santgar | SANT1553 | |
| SDS | Sigma-Aldrich | L3771 | |
| TEMED | Sigma aldrich | 102560430 | |
| TRI reagent | Sigma-Aldrich | T9424-200ML | monophasic solution of phenol and guanidinium isothiocyanate |
| Tris | US Biological | T8650 | |
| Tris base | Chem Cruz | sc-3715A |
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