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

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.