We developed a cell culture-based sensitive and quantitative assay that can be used to evaluate Sonic Hedgehog signaling, which is transduced mostly via the primary cilia. This assay can be used to examine genetic and epigenetic alterations, which are associated with primary cilia dysfunction. Despite the central role of primary cilia in Sonic Hedgehog signaling, current methodologies often lack the sensitivity required to assess the function of primary cilia under genetic alterations.
This protocol fills this gap by providing a cell culture-based assay, mimicking Sonic Hedgehog activation and Sonic Hedgehog target gene expression in ciliated cells. In comparison to other protocols, such as localization of smoothened protein along the primary cilia upon activation of Sonic Hedgehog pathway, our assay is quantitative, easy to follow, and sensitive as it is cell culture-based. To begin, remove the T25 cell culture flask containing near-confluent RPE-1 cells growing in five milliliters of complete medium from a 37 degrees Celsius and 5%carbon dioxide incubator.
Discard the culture medium from the cell culture flask and dislodge the cells with the enzymatic activity of a prewarmed 0.25%trypsin-EDTA solution. Resuspend the cells in a flask using a prewarmed complete medium. Count the cells on a hemocytometer to determine the cell density of the suspension.
Place two sterile 12-millimeter cover slips into two 35-millimeter cell culture dishes. Seed two times 10 to the power of five asynchronously growing RPE-1 cells in each dish containing cover slips. Add two milliliters of complete medium to each dish and grow the cells for 24 hours.
After incubation, remove the medium from the growing RPE-1 cells and wash the cells once with one milliliter of prewarmed DPBS buffer. Add two milliliters of prewarmed DMEM medium containing 1%penicillin streptomycin without FBS to each dish. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.
The next day, replace the medium with a serum-starved medium containing a smoothed agonist at a final concentration of 250 nanomolar. Incubate the cells for another 24 hours at 37 degrees Celsius and 5%carbon dioxide. After incubation, transfer the cover slips to a 24-well plate, placing one cover slip in each well.
Add 0.5 milliliters of chilled methanol to each well to fix the cells. Incubate the plate at minus 20 degrees Celsius for 10 minutes. Immediately wash the cover slips three times with 0.5 milliliters of wash buffer.
Discard the medium from the dishes. Add 0.5 milliliters of TRIzol Reagent directly to each dish. Distribute the Reagent evenly and let it stand for five minutes.
Pipette up and down a few times to create a homogeneous mixture and collect it in 1.5 milliliter, nuclease-free microcentrifuge tubes Add 0.1 milliliters of chloroform to each tube containing TRIzol-mixed cells treated with a smoothened agonist and DMSO. Incubate the tubes for two to three minutes before centrifuging at 12, 000g at four degrees Celsius. Transfer the aqueous phase containing the RNA to fresh microcentrifuge tubes.
Add 0.25 milliliters of isopropanol to the aqueous phase and incubate the tubes for 10 minutes. Centrifuge the samples at 12, 000g for 10 minutes at four degrees Celsius. Observe the formation of a white gel-like RNA pellet.
Carefully discard the supernatant using a micropipette. Then, resuspend the RNA pellet in 0.5 milliliters of freshly-diluted 75%ethanol. Briefly vortex the tubes to ensure mixing.
Centrifuge the samples at 7, 500g for five minutes at four degrees Celsius. Carefully discard the supernatant using a micropipette. Air-dry the RNA pellets for 10 minutes.
Resuspend the pellets in 25 microliters of nuclease-free water. Measure the total RNA concentration using a microvolume spectrophotometer at 260 nanometers. Use one to three micrograms of total RNA from each sample to synthesize cDNA following the manufacturer's instructions for a cDNA Synthesis Kit.
Set up the qPCR reactions in triplicate using one-is-to-five diluted cDNA from each sample with primer sets of GLI1, PTCH1, HHIP, SMO, and beta-actin genes, with a qPCR master mix in a multi-well, fluorescent-sensitive PCR plate. Cover the PCR plate with a suitable sealer. Place the plate in a qPCR instrument.
Run the standardized qPCR program with 40 amplification cycles, followed by a melting curve analysis. After the run, check the melt curve of each amplicon for a single peak at the desired temperature. Export the amplification data to a spreadsheet.
Calculate the relative expression of each transcript by normalizing against beta-actin expression, plot the relative expression data on a graph, and calculate the statistical significance of the changes in expression using an unpaired t-test. Most of the serum-starved cells assembled primary cilia with more than 85%ciliation. In this condition, the expression levels of GLI1, PTCH1, and HHIP were significantly increased in smooth agonist-treated cells compared to DMSO-treated cells, while SMO transcript levels showed no significant change.
