Cell Cycle Analysis: An Approach to Study Cell Cycle Regulation of miRNA-transfected Lung Cancer Cells

- 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