This method can help answer key questions in the RNA biology field about the mechanism and function of polarized RNA localization. The main advantage of this technique is that it allows for the real time visualization of endogenous RNA trafficking in live tissues. The implications of this technique extend toward the therapy of RNA-related diseases because it has a potential to uncover new therapeutic targets.
Though this method can provide insight into RNA processes in the fruit fly egg chamber, it can also be applied to other systems such as in zebrafish or culture cells. Generally, individuals new to this method will struggle because of the challenges associated with the design and delivery of the molecular beacon props. For molecular beacon microinjection, select the 40X oil objective and mount a coverslip with the dissected egg chamber onto the microscope stage.
Locate an egg chamber at the mid to late developmental stage that is properly oriented for microinjection. Then set up a microinjector with the appropriate injection and compensation pressures. Using the micro manipulator joystick, gently lower a needle loaded with one microliter of molecular beacon solution into the oil drop.
Bring the tip into focus toward the periphery of the field of view. Perform a clean function to remove the air from the needle tip and to ensure that there is flow from the needle and bring the needle to the home position. Focus on the egg chamber to be microinjected and bring the needle back into focus near the edge of the egg chamber.
Perform a fine adjustment of the z-position of the objective such that the membrane separating the follicle cells from the nurse cells is in focus. Insert the needle into a nurse cell for injection of the molecular beacons over a period of two to five seconds. To ensure reproducible microinjection success, focus carefully on the membrane separating the follicle cells and the nurse cells that will be injected while bringing the tip of the needle into focus with the micro manipulator.
When all of the molecular beacons have been injected, gently remove the needle and retract it to the home position. Change the objective to the desired magnification for image acquisition. Then focus on the egg chamber under the new objective and begin the image acquisition.
For spot detection of the molecular beacons, open the images in Image J.Use the convert to ICY tool to convert the images back to ICY. A scale bar will be automatically overlaid onto the stack. Edit the scale bar via the inspector window as necessary.
Then inactivate the eye icon for the scale bar under the layer tab to remove the scale bar from the original stack. Select detection and tracking, detection, and spot detector, and confirm current sequence input detection and channel zero are selected for the input and preprocessing parameters respectively. For detector, select detect bright spot over dark background using force use of 2D wavelengths for 3D only if there are not enough z-slices in the stacks to perform the analysis.
Select scales and sensitivity for each scale adding more scales for larger spots and confirm that region of interest from sequence is set for region of interest. For filtering, confirm that no filtering has been set and select the appropriate file format under output. If the spot detector results are to be used for tracking analysis, select export to swimming pool.
For colocalization analysis, repeat the spot detection for the other channel. To track the molecular beacon spots, select detection and tracking, tracking, spot tracking, and run the spot detector using the same parameters as for the spot detection. Click estimate parameters and select the desired target motion in the parameters estimation popup window.
Then click run tracking. To visualize the tracks, select detection and tracking, tracking, and track manager. For color track processor, select enable and select a color for the tracks.
From add track processor, select track processor time clip. In the check clipper window, enter the number of detections to be shown before and after the current time point. Save the track information as an XML track file and use the camera icon to obtain a screenshot of the results.
To project the stack along the z-direction, use the search bar to find the plugin and select maximum projection from the dropdown menu. In the inspector window, select the sequence tab, canvas, and rotation to adjust the image to the desired orientation and obtain a screenshot of the rotated image. Then select region of interest, 2D region of interest, choose region of interest shape, and select the region of interest on the image for cropping.
Install the timestamp overlay plugin and add a timestamp following the instructions in the popup window for directions on how to place and format the timestamp. To change or add the time interval, click edit in the sequence properties window. Activate the eye icon to add back the scale bar under the layer tab in the inspector window.
Then obtain a screenshot of the timestamped results and save the image in both TIFF and AVI formats. Using this method as demonstrated, it is possible to visualize the transport and localization patterns of endogenous mRNAs via molecular beacons at various stages of oogenesis and in particular at and after mid oogenesis. When individually injected into the same stage egg chambers, different oskar specific molecular beacons present the same pattern of localization.
Molecular beacons injected into the cytoplasm of a nurse cell will freely diffuse into adjacent nurse cells as well as into the oocyte. Thus, the beacons are able to hybridize with their targets and generate fluorescence signals at other sites than the microinjection site. In oskar GFP transgenic egg chambers at mid oogenesis, analysis of the acquisition data shows extensive colocalization for the fluorescence signal of genetically engineered GFP tagged oskar mRNA with the fluorescence signal detected using molecular beacons.
Moreover, 5D stacks can be further analyzed to determine oskar mRNA trajectories for long distance transport in both nurse cell and oocyte cytoplasm. After its development, this technique paved the way for researchers in the field of RNA biology to explore endogenous maternal mRNA transport and localization in the Drosophila egg chamber.
