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 identified^3,4,5,6,7.

In humans, basa....

Protocol

All procedures described here were approved by the Animal Ethics Committee of Cinvestav (CICUAL, # 0354/23). The laboratory animals were treated and handled in strict accordance with the journal's animal use guidelines and according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Ocular health before and after the procedures was evaluated by assessing ocular discharges, swollen eyelids, ocular abnormalities, and behavior changes.

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.......

Materials

Name	Company	Catalog Number	Comments
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