- A microRNA or miRNA is a small, non-coding RNA. Following mRNA transfection or introduction into a cell, it forms a miRNA protein complex. The complex targets and binds to specific mRNA and negatively regulates its expression, halting cell cycle progression.
To begin the analysis, add a suitable dehydrating fixative to a harvested culture of miRNA-transfected lung cancer cells. The fixative preserves the cells and also permeablilizes the cell membrane. Treat the cells to a mixture of propidium iodide, a fluorescent nucleic acid dye, and ribonuclease enzyme.
The dye enters the cells and stains both DNA and RNA. The enzymes specifically degrades the RNA allowing optimal DNA quantification. Use a flow cytometer to determine the proportion of cells halted in different cell cycle phases based on individual cells fluorescence intensity.
The cells in resting and growth phases have lower DNA content and hence, lower fluorescence intensity. The cells in the Gap 2 phase with double the amount of DNA produce twice the fluorescence intensity. The cells in the DNA synthesis phase induce a fluorescence value between the other two populations. The following protocol will analyze the cell cycle regulation of miRNA-treated lung cancer cells by measuring their DNA content using PI staining and flow cytometry.
- Transfect the cells as described in the earlier section of this video. Harvest cells from each sample in separate 15 milliliter sterile tubes by trypsinization. Next, wash the cells twice with 1X PBS by centrifugation at 751 g for 5 minutes and then remove the supernatant. Resuspend and break the pellet by adding 200 microliter ice-cold 1X PBS through pipetting.
Add 2 milliliters of 70% ice-cold ethanol dropwise to the tube while vortexing the tube gently to fix the cells. Incubate tubes for 30 minutes at room temperature and place them in 4 degrees Celsius for 1 hour. Remove the tubes from 4 degrees Celsius and centrifuge.
Add 2 mL ice-cold PBS and vortex and then remove the supernatant by centrifugation. Add 500 microliters of 1X PBS containing propidium iodide and ribonuclease A with concentrations 50 and 200 micrograms per milliliter, respectively, in each sample and incubate for 30 minutes at room temperature. Transfer samples into appropriate tubes protecting from light and run on flow cytometer.