This protocol shows a rolling circle amplification method to detect topoisomerase 1 activity in crude samples in a fast and simple way. The main advantages of this method are that it is easy to follow also by non-experienced researchers, and it is more sensitive compared to the other gel-based assays when crude extracts are used. The application of this protocol extend from the measurement of the response to drug treatment, to screening of small molecule compounds with potential anti-cancer or anti-infectious diseases effect.
Demonstrating the procedure will be Karol Mizielinski development specialist from our laboratory. To begin, attach a custom designed silicone isolator grid to a functionalized slide, thereby making the well maker. Press down on the silicone grid to avoid the formation of air bubbles between the surface of the glass and the silicone.
Next, prepare the primer mix by adding five micromolar, five prime amino primer in one time print buffer. Then add four microliters of this mixture to each well and incubate the well maker in a hybridization chamber with saturated sodium chloride. To generate closed circular substrates, add two microliters of five micromolar topoisomerase 1 specific substrate and two microliters of the recombinant topoisomerase 1 enzyme or a prepared cell extract to 16 microliters of onetime topoisomerase reaction buffer.
Incubate this circle mixture at 37 degrees Celsius for 30 minutes, and stop the enzyme reaction by adding two microliters of 1%sds. Immerse the well maker in a tray filled with buffer 1, preheated at 50 degrees Celsius. Pipette the buffer into the wells to ensure no air bubbles remain inside the well maker, and then incubate the tray for 30 minutes at 50 degrees Celsius.
After incubation, remove the buffer 1 and wash twice with distilled water with vigorous shaking for one minute between washes. Remove the water, next, add buffer 2, preheated at 50 degrees Celsius to the tray, then incubate and wash the well maker as demonstrated previously for buffer 1. Next, wash the well maker with 70%ethanol, with vigorous shaking for one minute and use compressed air to allow the setup to dry.
To perform hybridization, add four microliters of the prepared circle mixture to each corresponding well and place the well maker in a humidity chamber with distilled water at 37 degrees Celsius for one hour. At the end of incubation, wash the well maker for one minute each and buffer 3, buffer 4 and 70%ethanol, then let it air dry. Prepare and add four microliters of the RCA mixture to each well of the well maker, and incubate at 37 degrees Celsius for two hours in the humidity chamber.
To microscopically visualize the amplified fluorescent rolling circle products, first, wash the slide with buffer 3, buffer 4 and 70%ethanol. Dry the slide with compressed air and glue it onto a microscope slide. mark the position of the wells with a marker and then remove the silicone grid using tweezers.
To allow for visualization in the camera, mount the slide with two microliters of mounting medium without dapi and add a cover glass. Analyze the slide using a fluorescence microscope, with a 60x oil immersion objective lens and a camera. After imaging, import the pictures to image J and stack the images by clicking on Image, then Stacks and Images to Stack, then change the image type to 8 bit.
To set the threshold, click on Image, Adjust and then Threshold. Set the lower bar to 255 and adjust the upper bar so that only the right signals are red and the background is black. Sweep through the individual images and ensure they correspond to the images before adjusting the threshold.
Ensure the threshold is set as low as possible before the real signals start to disappear. To count the signals, click Analyze, followed by Analyze Particles and ensure that the summarized field in the image J settings is checked. Add four microliters of diluted horse radish peroxidase-conjugated anti-biotin antibody to each well and incubate at 15 to 25 degrees Celsius for 50 minutes in the humidity chamber.
At the end of incubation, wash the well maker three times with buffer 5 for three minutes each and let it dry. To visualize the chemiluminescence, add two microliters of freshly prepared ECL luminol and hydrogen peroxide mix to the wells and visualize the slide using a CCD camera or on x-ray films. Alternatively, to visualize with TMB, remove the silicone grid after the buffer 5 wash and add 400 microliters of TMB on top of the entire slide.
Keep the slide in a humidity chamber and wait for five to 10 minutes for color development. After a color development, wash the slide with 70%tethanol and photograph the slide with a camera or mobile phone for analysis with image J software. Import the picture to image J and change the image type to 8 bit by clicking on Image Type and then 8 bit.
Limit the measured area by using the rectangle drawing tool from the toolbar to measure the bands separately and draw the desired area. Measure the intensity by clicking, Analyze and then Measure. Next, move the originally drawn area to the next band and measure.
After measuring the intensity for all the bands, plot the data in the desired software. The Topoisomerase 1 activity as determined by a fluorescent scanner readout is depicted here, as evident from the quantification. This readout has a detection limit of 12.5 nanograms, representative images and the resulting quantification obtained using the fluorescent microscope readout, are shown here.
This readout method has a detection limit of 0.1 nanograms, the detection threshold of the enhanced chemiluminescence and the color o'metric readout methods was 6 nanograms. Using the same readouts, the detection limit was 1, 250 cells for the ECL and 312 cells for TMB. When topoisomerase 1 activity was measured from extracts of colorectal adenocarcinoma derived cells.
As an example of a drug screening application, the topoisomerase 1 activity was measured in the presence of camptothecin or dimethyl sulphoxide. The quantification results showed that camptothecin inhibits topoisomerase 1 one mediated circularization of the substrate as expected. This is a simple protocol that only requires specific attention to the washing steps and the reaction incubation times.
It is also possible to investigate how drugs inhibit topoisomerase 1 activity, using Reid is highly sensitive and easy to perform, it allows fast screening of potential topoisomerase 1 inhibitors, and the use of topoisomerase 1 activity as a biomarker for drug response in cancers.
