Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

Summary

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

Abstract

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Introduction

microRNAs (miRNAs) are short (19 to 25 nucleotides), naturally occurring, non-coding RNA molecules.Sequences of miRNAs are evolutionary conserved across species, suggesting the importance of miRNAs in regulating physiological functions¹. microRNA expression profiling has been proven to be helpful for identifying miRNAs that are important in the regulation of a variety of processes, including the immune response, cell differentiation, developmental processes, and apoptosis². More recently, miRNAs have been recognized for their potential use in disease diagnostics and therapeutics. For researchers studying mechanisms of ge....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Penn State University.

1. Assessment of the Estrous Cycle Stage

1. Properly restrain a female C57BL/6 mouse (8–9 weeks old) using the one-handed mouse restraint technique described in Machholz et al.¹⁰.
2. Fill the sterile plastic pipette with 10 μL of ultra-pure water.
3. Introduce the tip of plastic pipette into the vagina.
4. Gently flush the liquid 4–5 times to collect the sample.
5. Place the final flush containing vaginal fluid on a glass slide.
....

Results

The different cell types observed in smears are used to identify the mouse estrous cycle stage (Figure 1). These are identified by cell morphology. During proestrus, cells are almost exclusively clusters of round-shaped, well-formed nucleated epithelial cells (Figure 1A). When the mouse is in the estrus stage, cells are cornified squamous epithelial cells, present in densely packed clusters (Figure 1B

Discussion

MicroRNA profiling is an advantageous technique for both disease diagnosis and mechanistic research. In this manuscript, we defined a protocol to evaluate the expression of miRNAs that are predicted to regulate inflammatory genes in the lungs of female mice exposed to ozone in different estrous cycle stages. Methods for the determination of the estrous cycle, such as the visual detection method, have been described¹⁶. However, these rely on one-time measurements, and therefore are unreliable. To a.......

Materials

Name	Company	Catalog Number	Comments
C57BL/6J mice	The Jackson Laboratory	000664	8 weeks old
UltraPure Water	Thermo Fisher Scientific	10813012
Sterile plastic pipette	Fisher Scientific	13-711-25	Capacity: 1.7mL
Frosted Microscope Slides	Thermo Fisher Scientific	2951TS
Light microscope	Microscope World	MW3-H5	10X and 20X objective
Ketathesia- Ketamine HCl Injection USP	Henry Schein Animal Health	55853	90 mg/kg. Controlled drug.
Xylazine Sterile Solution	Lloyd Laboratories	139-236	10mg/kg. Controlled Drug.
Ethanol	Fisher Scientific	BP2818100	Dilute to 70% ethanol with water.
21G gauge needle	BD Biosciences	305165
Syringe	Fisher Scientific	329654	1mL
Operating Scissors	World Precision Instruments	501221, 504613	14cm, Sharp/Blunt, Curved and 9 cm, Straight, Fine Sharp Tip
Tweezer Kit	World Precision Instruments	504616
-80 ˚C freezer	Forma	7240
Spectrum Bessman Tissue Pulverizers	Fisher Scientific	08-418-1	Capacity: 10 to 50mg
RNase-free Microfuge Tubes	Thermo Fisher Scientific	AM12400	1.5 mL
TRIzol Reagent	Thermo Fisher Scientific	15596026
Direct-zol RNA MiniPrep Plus	Zymo Research	R2071
NanoDrop	Thermo Fisher Scientific	ND-ONE-W
miScript II RT kit	Qiagen	218161
Mouse Inflammatory Response & Autoimmunity miRNA PCR Array	Qiagen	MIMM-105Z
Thin-walled, DNase-free, RNase-free PCR tubes	Thermo Fisher Scientific	AM12225	for 20 μl reactions
miRNeasy Serum/Plasma Spike-in Control	Qiagen	219610
Microsoft Excel	Microsoft Corporation		https://office.microsoft.com/excel/
Ingenuity Pathway Analysis	Qiagen		https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/
R Software	The R Foundation		https://www.r-project.org/
Thermal cycler or chilling/heating block	General Lab Supplier
Microcentrifuge	General Lab Supplier
Real-time PCR cycler	General Lab Supplier
Multichannel pipettor	General Lab Supplier
RNA wash buffer	Zymo Research	R1003-3-48	48 mL
DNA digestion buffer	Zymo Research	E1010-1-4	4 mL
RNA pre-wash buffer	Zymo Research	R1020-2-25	25 mL
Ultraviolet ozone analyzer	Teledyne API	Model T400	http://www.teledyne-api.com/products/oxygen-compound-instruments/t400
Mass flow controllers	Sierra Instruments Inc	Flobox 951/954	http://www.sierrainstruments.com/products/954p.html