In this procedure, fluorescently labeled RNA. Transcripts are injected into XUS Cytes and their localization is monitored. First, the RNA is labeled with fluorescent nucleotides.
In an in vitro transcription reaction, the RNA is then loaded into a microinjection apparatus. The oocytes are placed on an injection dish and carefully micro injected. Finally, the oocytes are cultured to allow RNA localization to occur before imaging on the confocal microscope.
Hi, I'm James Gagnan from the laboratory of Dr.Kimberly Mallory at the Department of Molecular Biology, cell biology and biochemistry at Brown University. Today we're going to show you a procedure for visualizing subcellular RNA localization in Xap oocytes. We use this procedure in our laboratory to study the RNA architecture and protein factors required for RNA localization during early vertebrate development.
So let's get started. To begin this procedure, transcribe mRNA, which is both radioactively and fluorescently labeled as described in the accompanying written protocol. Add DS to degrade the template DNA, and then stop the reaction by adding EDTA.
Remove unincorporated nucleotides and other small molecules by spinning through a G 50 column. The RNA is then concentrated by ethanol precipitation, washed and resuspended. After determining the concentration by scintillation counting, dilute the RNA to 50 nano molar and store in five microliter aliquots at minus 80 degrees Celsius.
When ready to perform the microinjection thaw an Eloqua of the labeled RNA denature at 70 degrees Celsius for five minutes. Then spin for 10 minutes at maximum speed in a micro centrifuge at room temperature to remove particulates and keep on ice. Next, isolate the cytes trim pieces of xap post lavu ovary into a 50 milliliter conical two containing 25 milliliters of three milligram per milliliter.
Collagenase in a 0.1 molar potassium phosphate buffer at pH 7.4. Shake gently at 18 degrees Celsius due to batch to batch variation. Collagenase treatment time may vary and should be monitored carefully through visual inspection.
To ensure defoliation continue shaking for 15 minutes or until oocytes are visibly separated from ovary, allow the oocytes to settle to the bottom of the conical tube. Remove the solution and replace with MBSH buffer. Repeat this, wash twice more for a total of three washes.
Next, manually sort stage three to four CYTES and MBSH buffer under a standard light microscope. Stage three cytes are completely opaque, but white. While stage four cytes are slightly larger and speckled with pigment.
Cytes that are slightly transparent are too young and cytes that are fully pigmented or exhibit polarized pigment distribution are too old. Finally, prepare the needles for microinjection using three and a half inch capillaries pulling and beveling to make needles with an outer diameter of approximately 0.05 millimeters. Now that the mRNA oocytes and needles are ready, proceed to microinjection.
Start by calibrating the needle with desy water to deliver two nanometers per injection injection.Front. Load the needle using a gas controlled micro injector and calibrate the drop size using a micrometer after calibration front load the RNA to be injected into the needle. Next place the sorted oocytes on an injection dish In MBSH buffer, the dish contains a layer of black foam rubber.
The white cyte stand out well against a black background. Then carefully inject each oocyte with two liters of RNA at 50 nanomolar. Be sure to inject each oocyte only once and check the needle regularly for clogging when the injection is complete.
Expel the remaining RNA, rinse the needle with desy water and load the next RNA for injection. After the injection transfer the injected cytes to a well of a sterile 24 well plate up to a thousand. CYTES can be cultured per well.
Remove the buffer and replace it with 400 microliters of cyte culture medium per well. Then place the culture plate inside a plastic container with a wet paper towel to maintain a moist environment during culture, incubate oocytes at 18 degrees Celsius for time points ranging between eight and 48 hours after the desired incubation period. The oocytes can be prepared for microscopy after incubation of the injected oocytes.
Sort out any dead oocytes and place surviving oocytes in glass vials. Survival of larger than 90%of the oocytes is routinely observed. Replace the buffer with M-E-M-F-A fixative and rock the cytes for 20 minutes.
Protect the oocytes from sunlight during fixation after the fixation period is complete. Wash the oocytes by removing the fixative and replacing with an equal volume of MBSH buffer. Repeat this wash once more for a total of two washes.
Next, wash the oocytes into anhydrous methanol. First, remove half of the volume and replace with methanol. Repeat this two more times.
Following these washes, remove all of the solution and replace with methanol. Repeat this methanol wash one more time. The fixed and dehydrated oocytes can be stored at minus 20 degrees Celsius until ready to image.
Before imaging, add Marie's clearing medium to a glass bottom WPI fluoro dish to cover the imaging surface. Carefully transfer the oocytes from methanol to the fluoro dish, minimizing the volume of methanol transferred image the oocytes on an inverted confocal microscope, both a Zeiss LSM five 10 and aica TCSs P give good results. Now we'll show some representative images of subcellular RNA localization immediately following microinjection fluorescent RNA can be seen in the nucleus after eight hours of culture of fluorescently labeled control.
RNA can be seen uniformly distributed in the cytoplasm of the oocyte. However, a fluorescently labeled RNA containing sequences that recruit the transport machinery can be seen in the process of subcellular localization to the vegetable pole of the oocyte. We've just shown you how to micro inject fluorescently labeled RNAs into zap cytes and observe RNA localization by confocal microscopy.
When doing this procedure, it's important to use deci treated solutions to avoid RNAs contamination to culture, your cytes in OCM containing antibiotics, and to protect your cytes from sunlight whenever possible to avoid photo bleaching. So that's it. Thanks for watching and good luck with your experiments.
