This research was supported by grants from NIH K01HL133520 (PS) and K12HD055882 (PS). The authors thank Dr. Joanna Floros for the assistance with ozone exposure experiments.

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
<ol>
	<li>Properly restrain a female C57BL/6 mouse (8&#8211;9 weeks old) using the one-handed mouse restraint technique described in Machholz et al.10.</li>
	<li>Fill the sterile plastic pipette with 10 &#956;L of ultra-pure water.</li>
	<li>Introduce the tip of plastic pipette into the vagina.</li>...

<ol>
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The authors declare that they have no competing interests.

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...

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 gene regulation, measuring miRNA expression can enlighten systems-level models of regulatory processes, especially when miRNA information is merged with mRNA profiling and other genome-scale data3. On the other hand, miRNAs have also been shown to be more stable than mRNAs in a range of specimen types and are also measurable with greater sensitivity than proteins4. This has led to considerable interest in the development of miRNAs as biomarkers for diverse molecular diagnostic applications, including lung diseases.
In the lung, miRNAs play important roles in developmental processes and the maintenance of homeostasis. Moreover, their abnormal expression has been associated with the development and progression of various pulmonary diseases5. Inflammatory lung disease induced by air pollution has demonstrated greater severity and poorer prognosis in females, indicating that hormones and the estrous cycle can regulate lung innate immunity and miRNA expression in response to environmental challenges6. In this protocol, we use ozone exposure, which is a major component of air pollution, to induce a form of lung inflammation in female mice that occurs in the absence of adaptive immunity. By using ozone, we are inducing the development of airway hyperresponsiveness that is associated with airway epithelial cell damage and an increase in neutrophils and inflammatory mediators in proximal airways7. Currently, there are not well-described protocols to characterize and analyze miRNAs across the estrous cycle in ozone-exposed mice. 
Below, we describe a simple method to identify estrous cycle stages and miRNA expression in lung tissue of female mice exposed to ozone. We also address effective bioinformatics approaches to miRNA discovery and target identification, with an emphasis on computational biology. We analyze the microarray data using limma, an R/Bioconductor software that provides an integrated solution for analyzing data from gene expression experiments8. Analysis of PCR array data from limma has an advantage in terms of power over t-test based procedures when using small number of arrays/samples to compare expression. To comprehend the biological context of miRNA expression results, we then used the functional analysis software. In order to understand the mechanisms regulating transcriptional changes and to predict likely outcomes, the software combines miRNA-expression datasets and knowledge from the literature9. This is an advantage when compared with software that just look for statistical enrichment in overlapping to sets of miRNAs.

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>8 weeks old</td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10813012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-25</td>
			<td>Capacity: 1.7mL</td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>2951TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Microscope World</td>
			<td>MW3-H5</td>
			<td>10X and 20X objective</td>
		</tr>
		<tr>
			<td>Ketathesia- Ketamine HCl Injection USP</td>
			<td>Henry Schein Animal Health</td>
			<td>55853</td>
			<td>90 mg/kg. Controlled drug.</td>
		</tr>
		<tr>
			<td>Xylazine Sterile Solution</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td>10mg/kg. Controlled Drug.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td>Dilute to 70% ethanol with water.</td>
		</tr>
		<tr>
			<td>21G gauge needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher Scientific</td>
			<td>329654</td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>World Precision Instruments</td>
			<td>501221, 504613</td>
			<td>14cm, Sharp/Blunt, Curved and 9 cm, Straight, Fine Sharp Tip</td>
		</tr>
		<tr>
			<td>Tweezer Kit</td>
			<td>World Precision Instruments</td>
			<td>504616</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Forma</td>
			<td>7240</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum&#160;Bessman Tissue Pulverizers</td>
			<td>Fisher Scientific</td>
			<td>08-418-1</td>
			<td>Capacity: 10 to 50mg</td>
		</tr>
		<tr>
			<td>RNase-free Microfuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12400</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA MiniPrep Plus</td>
			<td>Zymo Research</td>
			<td>R2071</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Inflammatory Response &#38; Autoimmunity miRNA PCR Array</td>
			<td>Qiagen</td>
			<td>MIMM-105Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-walled, DNase-free, RNase-free PCR tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12225</td>
			<td>for 20 &#956;l reactions</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td>https://office.microsoft.com/excel/</td>
		</tr>
		<tr>
			<td>Ingenuity Pathway Analysis</td>
			<td>Qiagen</td>
			<td></td>
			<td>https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/</td>
		</tr>
		<tr>
			<td>R Software</td>
			<td>The R Foundation</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Thermal cycler or chilling/heating block</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR cycler</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipettor</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNA wash buffer</td>
			<td>Zymo Research</td>
			<td>R1003-3-48</td>
			<td>48 mL</td>
		</tr>
		<tr>
			<td>DNA digestion buffer</td>
			<td>Zymo Research</td>
			<td>E1010-1-4</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>RNA pre-wash buffer</td>
			<td>Zymo Research</td>
			<td>R1020-2-25</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Ultraviolet ozone analyzer</td>
			<td>Teledyne API</td>
			<td>Model T400</td>
			<td>http://www.teledyne-api.com/products/oxygen-compound-instruments/t400</td>
		</tr>
		<tr>
			<td>Mass flow controllers</td>
			<td>Sierra Instruments Inc</td>
			<td>Flobox 951/954</td>
			<td>http://www.sierrainstruments.com/products/954p.html</td>
		</tr>
	</tbody>
</table>

lung microrna profiling across the estrous cycle in ozone-exposed mice

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.

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</strong...

Watch this Scientific Journal Video about Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice at JoVE.com

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

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.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

This method can help answer key questions in the lung-immunity field about how to assess the expression of microRNAs that are predicted to regulate inflammatory genes within the lung. This technique offers the simple way for identifying the contributions of circulating hormones to lung microRNA expression. To assess the estrous cycle stage, manually restrain an eight to nine-week-old female mouse and introduce the tip of a plastic 10-microliter pipette filled with ultra pure water into the vagina.Gently flush four to five times to collect a sample, and deposit the final flush of vaginal fluid onto a glass slide. Then observe the unstained vaginal flush under a light microscope at 20X magnification. For ozone and filtered air exposures, place a maximum of four mice into one of two individual 1.2 liter glass containers with wire mesh lids.Put one glass container in an ozone chamber and one in a filtered air exposure chamber. Then adjust the ozone concentration to two parts per million, removing the container after three hours. Four hours after exposure, disinfect the exposed skin surface of the first experimental mouse with 70%ethanol and cut away the rib cage to expose the heart and lungs.Use forceps to submerge a 1.5 milliliter RNase-free micro centrifuge tube in the liquid nitrogen, and snap freeze the harvested lung in the liquid nitrogen. Then use a stainless steel tissue pulverizer to mascerate the whole lung, and split the pulverized tissue between two 1.5 milliliter microcentrifuge tubes. After harvesting and pulverizing the lungs from all of the animals in this same manner, add 500 microliters of guanidinium thiocyanate to each sample and sequentially homogenize each tissue with 18, 21, and 23 gauge needles.After the last homogenization, add 500 microliters of ethanol to each sample before vortexing for 15 seconds. Load the mixtures into individual spin columns in individual collection tubes for centrifugation and discard the flowthroughs. For DNase 1 treatment, add 400 microliters of RNA wash buffer to each column for another centrifugation and mix five microliters of DNase 1 and 75 microliters of DNA digestion buffer per sample in RNase-free tubes.Add the mix directly to the column matrices for a 15-minute incubation at room temperature, followed by two 400 microliter RNA prewash solution washes by centrifugation. To elute the RNA, add 35 microliters of DNase RNase-free water directly to each column matrix for a final centrifugation, and measure the total RNA concentration and purity of each sample on a spectrophotometer. Then store the RNA at minus 80 degrees Celsius.For retrotranscription of small RNase, add 200 nanograms of total RNA in 10 microliters of prewash solution to one tube of reverse transcription reaction mix per sample. After mixing, briefly centrifuge the samples and place them on ice until their incubation. Next, incubate the tubes for 60 minutes at 37 degrees Celsius, followed by a five-minute incubation at 95 degrees Celsius.At the end of the second incubation, dilute the CDNA with 200 microliters of RNase-free water per 20 microliter reverse transcription reaction, and add 10 microliters of each reaction mix sample to each well of a preloaded mouse inflammatory response and autoimmunity microRNA PCR array. The proestrus stage can be identified by the presence of round-shaped and well-formed nucleated epithelial cells. When the mouse is in the estrus stage, densely-packed clusters of anucleated, cornified, and squamous epithelial cells are observed in the smear.During metestrus, cornified epithelial cells and polymorphonuclear leukocytes are seen. In diestrus, leukocytes are generally more prevalent. RNA extracted from four mouse lungs, as demonstrated, exhibit nucleic acid concentrations ranging between 1198 and 2178 nanograms per microliter and average A260 to A280 ratio between 2.01 and 2.02, and an average A260 to A230 ratio between 2.139 and 2.223.In this table, log two-fold changes in microRNA expression between ozone and filtered air exposed mice are shown. After microRNA target filter and core analysis for 14 microRNAs, to obtain the significant expression log ratio and P values, these data can be mapped by the microRNA target filter. The core analysis also provides information about canonical pathways, diseases and function, regulators, and networks.Further, the functional analysis software can be used to produce a network analysis that shows the relationship between the microRNAs of interest and other molecules. While attempting this procedure, it is important to check the estrus cycle daily for at least three consecutive cycles prior to conducting an experiment. Following this procedure, other methods can be performed to answer additional questions about lung inflammatory gene expression across the estrus cycle.After it development, this technique paved the way for other researchers in the field of lung immunity to explore mechanisms of sex hormone regulation using other models.

lung-microrna-profiling-across-the-estrous-cycle-in-ozone-exposed-mice

Research

JoVE Journal

Immunology and Infection

6.0K visualizações.  Pennsylvania State University College of Medicine. Este método pode ajudar a responder perguntas-chave no campo da imunidade pulmonar sobre como avaliar a expressão de microRNAs que são previstas para regular genes inflamatórios dentro do pulmão. Esta técnica oferece a maneira simples de identificar as contribuições dos hormônios circulantes para a expressão de microRNA pulmonar. Para avaliar o estágio de ciclo estrous, contenha manualmente um rato fêmea de oito a nove semanas de idade e introduza a ponta de uma pipeta de plástic...