Herein we provide a thorough description of laser microdissection applied to achieve A compared RNA transcript profiling of silenced and uns silenced areas of the drosophila wing disc. This approach requires as biological material, manually dissected drosophila image discs as main equipment, a laser micro dissector. We use the LI A-L-L-M-D 6, 000 and a real time PCR machine.
We used a BioRad iq. Five small tools required are a concave plate, a small paintbrush microdissection forceps hanging drop slide insect pings, mounted on syringe needles, metal frame with pet membrane small plastic tubes. This method involves the following steps.
Step one to cross two suitable oph transgenic lines in order to trigger a localized and specific gene silencing in the progeny making use of the GAL four UAS system. Step two, to treat the dissection frames. Step three, to set up the laser micro dissector.
Step four, to manually collect the wing imaginal discs from the larval progeny of the genetic cross. Step five, to perform laser microdissection of specific areas of the GFP labeled imaginal discs. Step six, transcription profiling of equivalent areas of silenced and unsolid regions of the disc will allow the establishment of the effects triggered by silencing of your gene of interest.
Call it gene X in vivo transcription profiling will require extraction of RNA from the micro dissected areas, control of the accuracy of the manual dissection, evaluating GFP expression in the two samples by R-T-P-C-R, quantification of the expression of putative. Targets of the silence gene in the two samples by real time R-T-P-C-R or by performing a microarray analysis. Now more details on each step.
The first step, the genetic cross. The genetic cross involves GAL four UAS transgenic strains and is focused to obtain silenced imaginal discs in the larval progeny. The Versatile GAL four system allows you to trigger specific and localized gene silencing by RNA interference.
One strain introduces in the cross the driver transgene that expresses GAL four in a specific temporal or spatial pattern. And A-U-A-S-G-F-P reporter that allows the visualization of the GAL four expression domain. The second strain introduces a UAS responder transgene that expresses hairpin silencing RNA, able to specifically target gene X once expressed in the cell.
This RNA is cleaved to generate small interfering RNA, which is able to specifically silence the selected gene X in the progeny of this cross. The UAS silencing transgene is only transcribed in those cells expressing the GAL four protein, thus inducing a spatially restricted silencing of the X gene. Among the variety of applications, we focused on RNA transcript profiling in wing disc silenced by the Grailed GAL four driver, which directs specific silencing in the posterior compartment.
The silence region is marked by the expression of the U-A-S-G-F-P transgene. In the posterior compartment. Two events will be triggered expression of GGFP and silencing of gene x.
Step two, treatment of the dissection frames to inhibit RNAs activity. Wash the frame slides and dissection tools with DEPC water for at least one hour at room temperature and let them dry in a sterile chamber. To inhibit RNAs activity, use a brand new 50 ml tube already RNAs free, filled with DEPC water with forceps put in the tube, a hanging drop slide and a pet frame.
Both needed For the microdissection. Close the tube, Flip it upside down, then leave it to react for at least one hour. At room temperature, One hour later, move the DEPC water to another tube with the forceps.
Hold the slides Inside the tube so they will not fall. Then let the slides dry inside the tube. Step three, microdisect setup.
Firstly, turn on all the devices Needed, the fluorescence bulb, the microscope, and the software for the microdissection. A warning window will remind you to turn on also the laser. Do it and let the laser warm up while you finish the microdisect set up.
Now it is time to prepare the wells In which to collect the tissue.Cut. The Leica LMD 6, 000 laser cut equipment allows the loading of up to four tubes to collect different samples. For our purpose, we need just two tubes.
One, to collect the GFP positive silence Tissue, the other to collect GFP negative unile tissue With a micro pec. Fill both the tube caps with 20 microliters of tri reagent solution to Preserve the RNA. Now the microdisect is ready to use Step four, hand dissection of wing imaging discs from joof larvae.
Isolate wing discs from the larval progeny obtained as previously described, and be sure that other tissues do not contaminate the discs To achieve this goal, I dissection approach is quite different from that commonly used to collect the bulk of all original discs. First, wash the larva in ringer's solution to remove adhering media. Then transfer it Into a hanging drop slide and cut off the head immediately pulling the mouth down.
Using the insect pins now carefully dissect away other tissues. The red outline shows the imaginal wing discs to be collected. Keep the discs free from the adhering tissue quickly and gently Transfer each isolated wing disc to a dissection frame for immediate cut.
This procedure needs to be repeated until the total number of a hundred wing discs is reached. Step five, laser microdissection of specific areas of GFP labeled wing discs. Once the original wing disc is on the membrane, you can proceed with the cut.
Put the frame on its holder and fix the holder on the motorized stage. Look for the original wing disc using the LMD 6, 000 as a common microscope. Focus on it and set the cut.
Draw an oval area that will fit on both sides of the disc, the GFP positive and the other. Select the tube cap where you want to collect the GFP positive part. Push the Start cut button.
The micro dissector Proceeds with cutting the tissue with the speed and the power you set before by selecting the function move and cut. You can refine the cutting manually until the unselected Piece of tissue falls. The 3D animation shows what happens under the micros Dissector cut a laser being focuses on the tissue Making a cut along the selected perimeter.
Once the cut is done, the piece of tissue falls in the selected tube cap and consequentially in the tri Reagent. After the first Cut, move the cutting area on the other side of the wing disc to cut an equivalent GFP negative area. Before proceeding with the new cut, be sure to select a different tube cap to keep separate the GFP positive tissues from the GFP negative.
Push the start cut button after the automatic cut. Proceed to refining the cut manually as showed by the 3D animation before the second cut. The motorized stage moves the selected tube cap under the cut area.
The laser beam focuses on the tissue cut along the selected perimeter, and once the cut is done, the piece of tissue falls in the selected tube cap containing Tri reagent. To collect enough material, We repeated the procedure on a hundred imaginal wing discs. Once you have collected the samples, unload the tubes.
This is a delicate step that can make you lose all the work you've done so far. So be careful. Close the cap upside down and go ahead with the experiment.
This is the one with GFP positive tissue. This is the other one with GFP negative tissue. Step six, transcription profiling.
Spin the Tubes to detach the liquid from the cap and add Try reagent up to one milliliter. Perform RNA extraction following the standard try reagent protocol from a hundred areas of about 5, 000 micrometers squared each. Our yield was of 1.5 micrograms of RNA.
We use superscript three in vitrogen to synthesize CD NA with random hexa. Accuracy of the manual dissection was controlled by checking the GFP expression in the two samples by R-T-P-C-R. As showed in the gel with quantitative R-T-P-C-R.
We evaluated the expression levels of the silence gene X together with those of possible putative targets of the gene X mediated regulatory pathway. This diagram shows an example of the expression levels of gene X and one of its putative targets. Call them genes Y with respect to their basal expression levels in the anterior posterior compartments of unsolid wing discs.
The activity of the selected gene X was found reduced to 40%upon silencing. In contrast, one of its putative targets, gene Y was found significantly upregulated a sevenfold increase leading us to validate the hypothesis that it was negatively regulated by gene X.We conclude that the above described experimental approach can successfully be applied to the validation of putative targets on diverse regulatory pathways.
