The aim of this procedure is to obtain transcripts and metabolites from drosophila, heads in sufficient quality and quantity to perform consistent high throughput studies. This is accomplished by first generating and flash freezing flies of the appropriate genotype and age in triplicate. Next tissue is prepared for extraction by separating fly heads from the bodies while keeping tissue frozen.
Then the heads are lyophilized. Finally, the tissue is treated with triol or chloroform to extract transcripts or metabolites respectively. Ultimately, results can be obtained that show high quality RNA and metabolites can be obtained through bioanalyzer and NMR analysis.
Respectively, Though this method can provide insight and to the cellular basis of progressive neurodegeneration, it can also be applied to other diseases such as cancer, aging and stress mediated disorders. To begin make crosses by combining 10 virgins with a transgene of interest under the control of UAS and five temperature sensitive gal and waterless. Scale four males into large bottles.
Label the bottles and place them at 25 degrees Celsius After two days, clear the adults from the bottles and place the bottles at 18 degrees Celsius. When the progeny ELOs collect virgins and place them in small fly food vials, label the vials and place them at 18 degrees Celsius for 24 hours. After 24 hours at 18 degrees Celsius, shift the vials to 29 degrees Celsius.
Transfer the flies to clean vials every two days until they reach days 10 and 20. When the flies reach the desired age, count them and use a small funnel to transfer them into pre-labeled cryo vials. Invert the vials on the bench and wait 30 minutes.
Then flash. Freeze the vials by immersion in liquid nitrogen and store in a pre-labeled pre chilled freezer box. To collect fly heads, assemble the sieve and pre chill it at minus 80 degrees Celsius for at least 30 minutes.
Chill a nine by 14 centimeter piece of paraform on dry ice for five minutes. Empty the cryo vials onto the paraform a few at a time. Use a paintbrush to count the flies and store them in another cryo vial on dry ice until 100 flies are accumulated.
Next, dip the sieve in liquid nitrogen and then add the 100 flies to the upper chamber and shake the sve vigorously for one minute. Dip it in the liquid nitrogen again, and shake for an additional minute. Open the sieve.
Collect the heads from the lower chamber into a micro tube, and place it at minus 80 degrees Celsius until all the samples are ready to be lyophilized. Open the micro tubes, wrap the tops in para film and make small holes. Place the samples in the lyophilizer for a minimum of two days to extract the RNA.
Remove the samples from the freeze dryer and add three small sterile steel grinding balls to each micro tube. Place in the tissue grinder and run at 1, 500 strokes per minute. For one minute, add one milliliter triazole and use paraform to seal the tubes grind for one minute, tap the tubes upside down to dislodge the steel balls if necessary, and grind for an additional minute.
Pipette the mixture into a new RNAs free micro tube. Pellet the cellular debris in a tabletop micro centrifuge that 12, 000 times G for 10 minutes at four degrees Celsius. After transferring the S supernatant to a new micro tube, add 0.1 volumes of one three bromo chloro propane and shake vigorously for 15 seconds.
Incubate room temperature for three minutes, then spin at 12, 000 times G for 15 minutes At four degrees Celsius, transfer the top aqueous layer to a new micro tube. Precipitate the RNA by adding 0.5 volumes of ice cold isopropanol and invert six times to mix. Incubate the sample at room temperature for 10 minutes, then spin after removing the S supernatant.
Wash the RNA by adding one milliliter of ice cold, 70%ethanol and briefly vortex. Before spinning, remove the sup natant and air dry the pellet for at least 15 minutes. Add 80 microliters of DEPC water and place it at 55 degrees Celsius for 10 minutes.
Then store it overnight at four degrees Celsius the next day. Determine the RNA concentration after purifying 30 micrograms of bulk RNA according to the text protocol and determining the concentration, use the total RNA PICO assay on a bioanalyzer to assess the RNA quality for MNA purification. Place an RNAs free micro refuse tube containing 30 microliters of dyna beads on a magnet for one to two minutes, and remove the supernatant.
Re suspend the beads in 15 microliters of binding buffer. Incubate the tube on the magnet and remove the buffer after using DEPC water to adjust the volume of five micrograms of total RNA to 15 microliters. Heat the RNA at 65 degrees C for two minutes and place on ice.
Add 15 microliters of total RNA to the 15 microliters of pre-washed beads and incubate for 30 minutes of room temperature. Place on the magnet for one to two minutes and remove the supernatant. Once the RNA has been washed three times with 35 microliters of washing buffer B and the final wash removed, elute the MR.NA by adding 15 microliters of cold 10 millimolar tris hydrochloride and heating the sample to 80 degrees Celsius for two minutes.
Immediately transfer the S supernatant to an RN a's free tube and freeze at minus 80 degrees Celsius. The yield should be around one to 5%of the total RNA to perform methyl chloroform extraction. Begin by adding butylated hydroxy toluene to chloroform and to concentration of 0.1 milligram per milliliter.
Transfer 200 heads into chilled pre weighed conical screw cap Polypropylene tubes freeze at minus 80 degrees Celsius and lyophilize after determining the dry weight at five to seven zirconia beads and homogenizing a bead beater for two cycles of 20 seconds at 800 hertz. Using the dry weight, calculate the water methanol and chloroform to be added in the following ratios where X is the weight of the heavier sample in grams and the product is the volume of each liquid in milliliters to the tube containing the lyophilized heads. Add all of the methanol is calculated and 45%of the total water after homogenizing the sample in the bead beta for two more cycles of 20 seconds each add all of the chloroform and the remaining water to a conical glass vial on ice.
Transfer the bead mixture to the glass vial shake for 10 minutes, then incubate for 10 minutes on ice. Following a spin for five minutes at 2000 times G and four degrees C.Use a pasta pipette to remove the top polar phase and place it in a second micro tube. After freezing the lower nonpolar phase in a small glass vial at minus 80 degrees Celsius, dry it under a stream of nitrogen gas.
Add 620 microliters of derated chloroform to rehydrate the nonpolar phase. Transfer 600 microliters into labeled NMR tubes and place the polyphase in the sentri VA at 30 degrees Celsius. Run the Sentri VA until the tube is completely dry around seven hours.
Rehydrate the polyphase with 620 microliters of 0.1 molar sodium phosphate buffer in deuterium oxide with one millimolar TMSP as an internal standard and vortex for 30 seconds. Following a spin at 10, 000 RPM and four degrees Celsius for five minutes. Transfer 600 microliters of the SUPINATE to NMR tubes to determine the expression dynamics of the expanded at attacks in three trench gene under the control of GAL A TTS and DOLA scale four Western blots were performed from different time points at the restrictive temperature and as shown here, expression was faint at six hours and continued to increase until 48 hours where it reached saturated levels.
The protein was also detected using immunofluorescence in dissected fly brains first at 24 hours, then reaching maximal levels between 48 and 72 hours after the temperature shift to determine the toxicity of ATIN 3 78 Q, we collected flies expressing LAX Z aax in 3 27 Q and AAX in 3 78 Q and recorded their longevity. Whereas flies expressing lack Z and ATEX in 3 27 Q displayed over 90%viability after 20 days flies expressing atex in 3 78 Q showed low survival at day 20. In this example, two samples of total RNA before DNA's treatment were run on a bioanalyzer chip.
The high quality RNA sample one produced three sharp RNA bands two just below 2000 nucleotides in size, and a smaller band below 200 nucleotides representing the ribosomal RNAs. These qualities were also observed in the bioanalyzer traces. In contrast, a degraded RNA sample sample two did not produce the sharp peaks of the ribosomal RNAs and accumulated multiple bands of less than 500 nucleotides.
This size distribution was readily distinguished in the bioanalyzer traces in which broad peaks of shorter size were noted. Only RNA displaying maximum quality is used to generate libraries. The NMR analysis in this figure identifies a few prominent peaks corresponding to highly abundant metabolites, such as glucose and sucrose, and two key metabolic acids, lactate and acetate according to chemical shifts in the literature.
After watching this video, you should have a good understanding at how to obtain high quality transcripts and metabolites from large collections of Joss heads that will enable you to uncover the molecular and cellular perturbations induced by toxic amyloids that mediate neuronal cell loss.
