Here we applied microinjection of fluorescently labeled RNAs to detect RNA sequences which are essential for localization of short nuclear RNAs into nuclear structures called Cajal bodies. This technique has two major advantages. First, it can be applied for short RNAs which are otherwise difficult to label and track.
And second, it allows rapid screening of different sequences and testing their role in RNA localization. One day before microinjection, seed HeLa or other adherent cells on 12 millimeter coverslips to reach 50%confluency at the time of microinjection. To begin, place a prepared coverslip into the center of a Petri dish, add cells and two millimeters of culture media.
The following day, place the cell filled Petri dish onto a microscope stage. Using a microloader, load three microliters of the snRNA mixture into the needle and install the needle into the holder of the microinjector at an angle of 45 degrees with respect to the surface of the Petri dish. Use a 10X long distance objective to find the needle.
First, set the speed coarse on the injector and lower the needle until it touches the culture medium. Using the microscope binoculars, find the bright spot which is the place where the needle touches the culture medium. Change the speed to fine and further lower the needle while looking into the microscope until the tip of the needle is observed.
Move the needle in the middle of the visual field and switch to a 40X long distance objective. After this, select the cell and move the needle above the cell. Set the pressure and time for the cell contact.
Move the needle down into the cell and then set the lower limit of the micromanipulator. Setting the lower limit is the most critical part of the protocol. Because coverslip surface is not perfect and because each cell is different, you will have to set the lower limit several times during the injection session.
Next, move the needle back above the cell and press the injection button on the joystick of the injector. The needle will move automatically inside the cell to the place where the limit is set to inject the RNA mixture. To finish, push the menu button on the injector and remove the needle from the holder.
Then disconnect the tube from the injector before switching off the injector and the micromanipulator. After the microinjection, return the cells to a carbon dioxide incubator and incubate at 37 degrees Celsius for one hour. Next, rinse the cells three times with PBS at room temperature.
Fix the cells for 20 minutes with 4%paraformaldehyde in 0.1 molar pipes at pH 6.9. Then wash the cells three times with room temperature PBS. If only snRNA localization is analyzed, briefly rinse the cells in water and mount in a mounting medium with DAPI.
In case of additional protein localization, permeabilize the cells with 0.5%Triton X-100 in PBS for five minutes at room temperature. Rinse the cells three times with PBS and stain them with the appropriate primary and secondary antibodies as outlined in the text protocol. Then rinse three times with PBS and rinse once in water.
After this, mount the cells in a mounting medium with DAPI. When ready to image, use a high-end fluorescence microscopic system equipped with an immersion objective to acquire the images. Once the microinjected cells are identified, collect a stack of 20 Z sections with 200 nanometer Z steps per sample and subject to mathematical deconvolution.
To quantify the fluorescent signal, first open the microscopy image in ImageJ and split channels into windows. Synchronize windows by the synchronize window tool. Next, draw the region of interest around a Cajal body identified by coilin immunostaining.
Measure the intensity of the snRNA signal in the coilin-defined ROI. Determine the intensity of snRNA fluorescence in ROI randomly placed in the nucleoplasm. Save measured values and calculate the ratio of RNA signal in the Cajal body and nucleoplasm.
In this study, fluorescently labeled snRNAs are microinjected in order to monitor their transport inside the cell. To test whether the SM site is important for Cajal body accumulation, snRNA lacking the SM site is microinjected directly into the nucleus. Injection into the cytoplasm serves as a control while full length snRNA is microinjected to serve as a positive control.
After incubation, microinjected cells are fixed and the Cajal body marker coilin is visualized by indirect immunofluorescence. The full length snRNA accumulated in Cajal bodies after both nuclear and cytoplasmic injections. In contrast, snRNA lacking the SM site remained in the cytoplasm after microinjection into this compartment while the nuclear microinjection of snRNA without the SM site reveals that the SM binding sequence is important for Cajal body localization as this snRNA remains in the nucleus but does not accumulate in Cajal bodies.
Sometimes the microinjection into only one cellular compartment fails and tricked Dextran 70 kilodalton can be found in both the nucleus and the cytoplasm. These cells are discarded from further analysis. Despite the fact that the fluorescently labeled snRNAs are stored at negative 80 degrees Celsius before the injection, RNA degradation can occur.
Degraded snRNA injected into the cytoplasm is not transported into the nucleus and stays localized in the cytoplasm. Such snRNA should be discarded and new snRNA should be synthesized. It is important to correctly adjust injection and compensating pressures for your cell type.
And as said before, setting correct lower limit for cell injection is essential for a good microinjection. Please remember that the phenol-chloroform used during RNA isolation is dangerous and that the microinjection needle is a sharp object. Be careful when handling these materials.
