This procedure describes an optimized fish method to assess expression and localization features of RNA in whole mount Drosophila embryos. This is accomplished by first synthesizing a labeled RNA probe, complimentary to an Mr NA or non-coating RNA of interest by in vitro transcription of an appropriately selected DNA template. In parallel fly population cages are used to harvest, fix and perme embryos of the desired developmental stage.
After several post fixation steps, the embryos are hybridized with the labeled antisense RNA probe. Finally, following extensive washing of the samples, the hybridized probe is detected by immuno labeling with a specific antibody and fluorochrome conjugated detection reagents, thus providing a sensitive readout of the target RNA distribution. Ultimately, the results yield highly resolved fluorescent signals that are analyzed by standard fluorescence microscopy in order to document the spatial and temporal expression features of the target RNA during drosophila embryogenesis.
The main advantage of this technique over existing methods such as Northern blot or R-T-P-C-R is one can assess the RNA expression within intact tissue specimen. While this method is intended for the analysis of RNA expression properties in early drosophila embryos, it can also be employed for the analysis of embryos at later developmental stages of tissue dissected from larvae or adult flies. An important aspect of our procedure is upscaling in sample number that we can achieve by performing fish in PCR place, which substantially increases throughput of the approach.
The members of the lab demonstrating this procedure include undergraduate research resistance, graduate students, and post-doctoral fellows. Begin with well-fed population cages. Replace the plate with an apple juice yeast plate and leave the flies for an hour at 25 degrees Celsius.
Then replace the apple juice yeast plate to harvest early stage embryos. Incubate the cage at 25 degrees Celsius and wait until the desired developmental stage of the embryos has been reached while waiting in a chemical hood, mix the chemicals to make 37%formaldehyde in a glass inhalation vial. Boiled the solution until the PFA powder is completely dissolved.
Then cool it to room temperature and filter it into a new scintillation vial. Next, prepare a biphasic fixation solution. Combine PBS with 37%formaldehyde in a scintillation vial, and then add heptane when ready.
Harvest the embryos from the apple juice plate with room temperature, tap water, and delicate sweeping with a fine paintbrush. Transfer the resuspended embryos into a basket and rinse them with lukewarm tap water. Now dec coate the embryos for 90 seconds by bathing the basket in a fresh 3%bleach solution.
Then remove the bleach with lukewarm tap water until the bleach smell is gone. To collect the embryos, disassemble the collection basket and gently transfer the embryos into the biphasic fixation solution. Using the paintbrush, the embryos will accumulate at the interface between the PBS and heptane layers.
Now seal the scintillation vials, fasten them to a vortex mixer and shake them for 20 minutes on the lowest setting. After the shaking, discard most of the lower PBS and upper heane phases using a pasture pipette. Then aspirate the remaining mixture into the pipette and carefully drop out the lower phase without losing the embryos.
Deposit the embryos into a 1.5 milliliter tube containing a biphasic solution of 500 microliters of heptane and 500 microliters of methanol. Shake the tube vigorously for 30 to 45 seconds to crack the patella membranes. Once cracked, the embryos will sink into the methanol.
Discard the upper phase and uncracked embryos and add one milliliter of methanol. Now briefly shake the tube for five seconds and wash the embryos three times with methanol. After the washes, the embryos can be stored for several months at minus 20 degrees Celsius.
Begin by adding 500 microliters of PK to each of embryos and incubate the samples for 10 minutes at room temperature without shaking. Take great care to damage the samples by over digesting them with protein a k, or through sloppy manipulation. During the PK incubation, mix the embryos every two minutes by jetting them with a pipette.
After 10 minutes, put the embryos on ice for an hour. After an hour, remove the PK solution at room temperature. Wash the embryos twice with glycine in PBT for two minutes.
Now, fix the samples for 20 minutes in one milliliter of 3.7%formaldehyde in PBT with rocking. Wash out the formaldehyde with five rinses in PBT. Then rinse the embryos with one milliliter of equal volumes PBT and HI solution, and replace the mixture with 0.5 to one milliliter of pure solution.
At this point, the embryos can be stored for several days to weeks at minus 20 degrees Celsius. In a 96 well plate aliquot 10 to 15 microliters of settled embryos per well. Use a wide bore tip to avoid damaging the embryos.
Now prepare the pre hybridization solution by boiling 100 microliters of hype solution per sample. Well for five minutes, cool the Pre-Hype solution on ice for five minutes, and then add 100 microliters of it to each plate well, which contains the embryo samples. Retrieve the prepared RNA probe quantified by a NanoDrop spectrophotometer.
Also, to verify the probe's quality, run one to two microliters of it on an agro gel. Now for each sample, dilute about 100 nanograms of probe in 100 microliters of HI solution. It is vital to work under RNAs free conditions.
When handling the probe. Heat the solution to 80 degrees Celsius for three minutes. Then cool it on ice for at least five minutes.
It can be kept cooled for several hours. Retrieve the plate and remove the Pre-Hype solution from the embryos by aspiration. Then add 100 microliters of hype solution with the RNA probe to each.
Well hybridize the probe at 56 degrees Celsius for 12 to 16 hours the next day. A series of five solutions are prewarm at 56 degrees Celsius. The first is pure hype solution, and the last is pure PBT.
The other three are dilution of HI into PBT. Once warmed quickly rinse the samples once in pure hype solution. Next, perform three 15 minute washes, beginning with the least diluted hype solution followed by each of the successive dilutions.
Lastly, wash the samples four times for five minutes in pure PBT. Then bring the samples to room temperature. Now proceed with antibody applications to detect the probe to improve sample mixing.
During these steps, the plate can be placed in a small box that is attachable to a mutating mixer using the described protocol, the classic pair rule gene runt was analyzed at distinct stages of embryogenesis. Fish analysis revealed how the initial broad expression in the mid portion of the embryo at embryonic stage four is refined into a segmented stripe pattern. In later stages, fish was performed using various diggen and labeled antisense, RNA probes hybridized to zero to four hour old drosophila embryos.
The hybridized probes were detected by sequential incubations with a biotinylated antigen and antibody strep avid in HRP and Ceramide. The samples were mounted and analyzed by fluorescence microscopy. A broad mosaic of fish results were observed in these stage four to seven embryos While attempting this procedure, it's important to use precautions at various steps such as protecting your RNA probes and degradation by working under RNAs free conditions and using RNAs free supplies.
Once this basic is mastered, additional steps can be added to the method to do multi labeling experience in order to visualize different RNAs or specific protein markers in the same sample. After watching this video, you'll have a good idea of how to perform fluorescent in C two hybridization in order to document the expression or subcellular localization dynamics of your favorite RNA during Drosophila embryogenesis Happy Fishing.
