The overall goal of this procedure is to fluorescently label and quantify the amount of nascent RNA produced under treatment conditions using quantitative imaging analysis to accomplish this. Cultured cells plated on cover glass are treated with the alkein modified base. Five etal uridine referred to hereafter as five EU and incubated under hypoxic conditions.
During the incubation, five EU is incorporated during RNA synthesis without interrupting the cellular transcription machinery. Next, the incorporated five EU is then fluorescently labeled by performing a copper catalyzed azide aine cyclo edition or click reaction. The fluorescently labeled nascent RNA is then imaged by fluorescence microscopy and the resulting images are analyzed to compare the amount of RNA generated under treatment and control conditions.
Statistical analysis of the resulting data shows the changes in nascent RNA generation in response to hypoxia through quantification of five EU incorporation and detection by fluorescence microscopy. The main advantage of this technique over existing methods like quantitative BCR, is that it allows direct visualization of RNA production in cells at the single cell level. Although here we use this method to study how cells respond to low oxygen, it can also be used to study responses to chemotherapeutic drugs.
Our responses to additional stress responses demonstrating a procedure will be John Leston, a graduate student from my laboratory with help from ish Kova, also a graduate student, and Humana Druker, a postdoctoral fellow. Finally, Gus Ferguson will demonstrate the use of image visualization software. Begin this experiment by dipping several cover slips in 70%ethanol to sterilize them.
Place the cover slips on the edge of a plate in the tissue culture hood and allow them to air dry under laminar flow. Once the cover slips are dry, place one in each of six 3.5 centimeter dishes. Add two milliliters of warm complete DMEM to each dish.
Press down with forceps to ensure that each cover slip remains adhered to the bottom of the well. Next to detach the U2 OS cells from the tissue culture dish Wash once with three milliliters of PBS. Then apply four milliliters of warm 0.05%trypsin, 1%EDTA and incubate at 37 degrees Celsius for seven minutes.
After seven minutes have passed Resus, suspend the cells in six milliliters of warm complete DMEM to inactivate the trypsin EDTA. Count the cells using a hemo cytometer seed each of the 3.5 centimeter dishes containing cover slips with two times 10 to the fifth cells. Gently rock the dish to ensure an even cell distribution incubate overnight at 37 degrees Celsius, 21%oxygen, 5%carbon dioxide to allow the cells to adhere.
The next day, remove five of the dishes from the incubator. Leave the remaining dish undisturbed. It will be used as the normoxic positive control.
Place four of the dishes into the hypoxia workstation. Expose the cells to hypoxic conditions for 75 minutes, 90 minutes, two hours, and 24 hours. To the other dish.
Add acton mycin D to the medium to a final concentration of 10 micrograms per milliliter and place it back in the 37 degree incubator under normal oxygen conditions for four hours. This dish functions as the normoxic negative control. While the cells are incubating, prepare and gather the following reagents in preparation for the click it assay.
The click it assay kit PBS at a pH of 7.2 to 7.6 3.7%Paraform aldehyde in PBS 1%Triton X 100 in PBS deionized water dimethyl sulfoxide 10 molar sodium hydroxide, 37%hydrochloric acid and pH indicator strips. You should plan to be ready to commence a labeled an assay undertreated conditions. One hour before the hypoxia treatment ends To label the cells prepare a two x working solution of EU from the 100 millimolar stock in prewarm complete medium.
Next, add an equal volume of two XEU to the medium in the dish containing the acton mycin D treated cells, thereby diluting the EU to the working concentration. Then in the hypoxia chamber, add the EU stock to the hypoxia treated cells. Incubate for one hour under treatment cell culture conditions after the hypoxia treatment or after four hours for the controls.
Wash each dish once with PBS and add one milliliter of the 3.7%Paraform aldehyde stock under treatment conditions, incubate for 15 minutes under treatment conditions. Following fixation, remove the cells from the hypoxia chamber and place them in the fume hood. Pour the fixative off taking care to dispose of the para formaldehyde waste responsibly.
Then wash the cells once with PBS. Perform the next steps at room temperature, keeping all solutions on ice at two to six degrees Celsius. Next to perme the cells, remove the wash solution and add one milliliter of 1%tritton X 100 in PBS to each, well incubate for 15 minutes at room temperature.
Remove the permeation buffer and wash each well once with PBS. Remove the wash solution from the cells and add 500 microliters of freshly prepared RNA imaging kit reaction cocktail to each sample. Protect the cells from light and incubate for 30 minutes at room temperature.
After the incubation, remove the RNA imaging kit reaction, cocktail, and wash. Once with one milliliter of RNA imaging kit reaction, rinse, buffer, any additional antibody labeling should be performed at this time. To stain the DNA, wash the samples with PBS, then dilute the herst 3, 3, 3, 4, 2 to one to 1000.
In PBS, add one milliliter of the diluted HERC solution to each. Well protect from light and incubate for 15 minutes at room temperature. After the incubation, remove the HERC solution and wash the cells twice with PBS to mount the cover slips.
Place 10 microliters of mounting medium on the center of a standard microscope. Slide and place the cover slip cell side down on the drop of mounting medium. Avoid generating bubbles as this will obscure subsequent image acquisition.
Apply clear nail varnish to the perimeter of the cover slip to fix it to the microscope slide. Allow the varnish to dry for five minutes at room temperature. Protected from light samples may be stored at negative 20 degrees Celsius.
Following mounting, acquire images using a wide field microscope capable of fluorescence detection. Perform imaging using a 40 V 1.30 NA oil immersion lens and capture images with a cooled CCD camera image. A minimum of 30 cells following image acquisition.
Use image processing software to devolve the images. This will correct for optical distortion by applying a software based mathematical formula to the captured images. The result is a clearer image that will ensure the subsequent analysis is limited to the imaged cells only.
Next, import the images into the Omer O client. Thumbnails of all the images obtained will be displayed next to normalize the rendering settings for all images in the same experiment. Apply the rendering settings from control normoxic cells to all other conditions using the region of interest ROI tool in the analysis software.
Select nuclei from each image in the software. Choose the intensity analysis option and obtain mean intensities for each ROI for 30 to 50 cells. Save the data and then calculate the average and standard for each condition.
Finally, using data graphing software constructs scatterplots of all individual mean intensity values to reflect the variability between cells under each condition. Plotting intensity data in this way is useful because it allows a rapid comparison of treatment results and clearly demonstrates significant heterogeneity within a treatment cell population that's otherwise obscured using standard data representation. Alternatively, construct bar graphs to represent average mean intensity plus standard deviation of nascent RNA to quantitatively determine total RNA synthesis U2 OS cells treated with hypoxia and cultured in the presence of five al uridine or EU were then processed and analyzed.
As demonstrated in this video, fluorescence intensities from all cells within each treatment population were averaged and plotted. In this bar chart, a student's T-test was used to calculate the statistical probability of each treatment group differing significantly from the control. Interestingly, these data show that there is a statistically significant increase in global RNA synthesis over time.
When U2 OS cells are treated with hypoxia for longer than an hour and a half as expected, ACTO mycin D treatment completely ablates the fluorescence intensity. These data suggest that following longer periods of hypoxia treatment, the cell focuses its efforts on the production of RNA, even in this hostile hypoxic environment. After watching this video, you should have a good understanding of how to label and quantify nascent RNA at the single cell level in hypoxia and normoxia once mastered, this technique can be done in two hours following this procedure.
Other methods like co staining with antibodies for RNA polymerase and HIF one can be performed to determine whether RNA polymerase levels are altered and whether HIF is activated. Don't forget that working with formaldehyde can be extremely hazardous while performing this procedure. Always be sure to wear gloves and work in a fume hood.
