Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
May 13th, 2014
We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.
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.
Overview
This article describes a novel technique for analyzing global RNA synthesis in hypoxic conditions using imaging. The method employs click-chemistry labeling of RNA, enabling visualization of RNA changes at the single-cell level, which complements traditional averaged RNA techniques.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- RNA Analysis
Background
- Understanding RNA synthesis under hypoxia is crucial for studying cellular responses to low oxygen.
- Existing methods often average RNA data, obscuring individual cell responses.
- Click-chemistry labeling allows for direct visualization of RNA production.
- This technique can also be applied to study responses to chemotherapeutic drugs.
Purpose of Study
- To fluorescently label and quantify nascent RNA produced under hypoxic conditions.
- To visualize cell-to-cell changes in RNA synthesis.
- To improve upon existing RNA analysis techniques.
Methods Used
- Cells are treated with an alkein modified base and incubated under hypoxic conditions.
- Incorporated RNA is fluorescently labeled using a click reaction.
- Fluorescence microscopy is used to image the labeled RNA.
- Statistical analysis is performed to compare RNA generation under different conditions.
Main Results
- The technique allows for direct visualization of RNA production at the single-cell level.
- Statistical analysis reveals significant changes in RNA synthesis in response to hypoxia.
- The method demonstrates advantages over traditional quantitative techniques.
- Potential applications extend to studying various stress responses in cells.
Conclusions
- This novel approach enhances the understanding of RNA dynamics under stress conditions.
- It provides a powerful tool for researchers studying cellular responses to hypoxia and other treatments.
- The method's versatility allows for broader applications in cellular biology research.
Frequently Asked Questions
Executive Industry Relevance
This method enables direct visualization of nascent RNA synthesis at single-cell resolution under hypoxic conditions, addressing a key limitation of population-averaged assays that obscure heterogeneous cellular responses. By quantifying temporal and cell-to-cell variability in RNA production, it supports mechanistic de-risking in target validation and improves predictive confidence in preclinical models of hypoxia-driven diseases such as cancer and ischemia. The approach enhances translational continuity by linking molecular phenotypes to functional outcomes in disease-relevant systems.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of transcriptional heterogeneity in hypoxic microenvironments, clarifying pathway activation and therapeutic target engagement.
- Operational Value: Provides quantitative, imaging-based readouts for assessing target modulation in single cells, reducing reliance on bulk population averages.
- Predictive Value: Supports phenotypic screening by identifying subpopulations with differential RNA synthesis responses to hypoxic stress or compound treatment.
Screening & Assay Development
- Scientific Value: Generates standardized, reproducible measurements of nascent RNA incorporation via EU labeling and click-chemistry detection.
- Operational Value: Compatible with fluorescence microscopy and image analysis pipelines, enabling scalable, multiplexable assay formats for compound screening.
- Assay Readiness: Produces quantifiable fluorescence intensity data that can be normalized and statistically analyzed to compare treatment effects across conditions.
Translational & Preclinical Research
- Disease Relevance: Models hypoxic stress in cancer cells (U2 OS), a system relevant to tumor microenvironment studies and angiogenesis-targeted therapies.
- Translational Continuity: Connects early transcriptional responses to downstream phenotypic adaptations, supporting risk-adjusted advancement decisions in preclinical development.
- Mechanistic De-risking: Reveals sustained RNA synthesis under prolonged hypoxia, indicating adaptive transcriptional programs that may influence therapeutic resistance or survival pathways.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing through lead identification to preclinical validation, particularly for targets modulated by hypoxic signaling or metabolic adaptation.
- Discovery Biology: Facilitates hypothesis testing of oxygen-sensing pathways and transcriptional regulators by visualizing real-time RNA synthesis dynamics at single-cell resolution.
- Screening: Enables assay development for compounds that alter hypoxic response, with quantitative imaging outputs supporting hit validation and structure-activity relationship studies.
- Analytics: Generates mean fluorescence intensity and standard deviation data per condition, allowing statistical comparison (e.g., student’s T-test) of RNA synthesis changes across treatment groups and time points.
- Translational Research: Links hypoxia-induced transcriptional activity to cellular adaptation in disease models, supporting biomarker alignment and mechanistic insight in preclinical workflows.
- Enterprise Reuse: Establishes a reusable imaging-based platform for assessing transcriptional responses to diverse stressors, including chemotherapeutic agents and ischemic conditions.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by revealing cell-to-cell heterogeneity in RNA synthesis, reducing false negatives from averaged data.
- Operational Value: Standardizes RNA synthesis measurement via click-chemistry labeling and fluorescence microscopy, improving reproducibility across labs and experiments.
- Strategic Value: Informs go/no-go decisions by identifying subpopulations with sustained transcriptional activity under stress, indicating potential resistance mechanisms.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on their influence on hypoxic transcriptional programs and adaptive responses.
Implementation Considerations
- Requires expertise in cell culture, hypoxia workstation operation, and fluorescence microscopy imaging.
- Dependent on click-chemistry reagents (EU, azide dyes, copper catalyst) and compatible imaging systems with appropriate filter sets.
- Necessitates standardized fixation, permeabilization, and labeling protocols to ensure consistent signal detection across samples.
- Requires adaptation of incubation times and EU concentrations when applying to different cell types or stress conditions beyond hypoxia.
- Limited by the need for specialized hypoxia-controlled incubation equipment and careful handling of formaldehyde-based fixatives.
