Laser-capture microdissectie van Human Prostatic Epithelium voor RNA-analyse

The overall goal of this laser capture micro dissection protocol is to isolate total RNA from pure populations of prostatic epithelial cell types from heterogeneous frozen prostate tissues for downstream gene expression analyses. This method can help analyze patient specimens and answer key questions in the prostate cancer research field, such as how RNA expression differs in benign and disease epithelial prosthetic tissue samples, or in clinical trial specimens. The main advantage of this technique is that laser capture micro dissection enables RNA isolation of homogeneous populations of epithelial or str cells from frozen prostate tissue.

Generally, individuals new to this method will struggle to acquire high quality RNA because they do not know which steps are critical to maintain RNAs. Free conditions Begin by gathering all reagents and tools needed for the procedure. After allowing the frozen prostate tissue to equilibrate inside the cryostat for 30 minutes, squirt mounting medium into the bottom of the cassette and quickly mount the tissue using chilled forceps.

Place the cassette into the chiller of the cryostat for five minutes to set the mounting medium. Once set, squirt frozen, mounting medium onto the chuck and carefully press the mounted tissue into it. Bottom side up.

Allow the preparation to set for five minutes. Before placing the chuck into the holder, lock the handle into position and insert a new blade onto the cryostat. After unlocking the handle, carefully advance the sample until the desired tissue exposure is reached.

When ready, adjust the cryostat to five microns and cut one or two sections. Place these sections onto a charged glass slide for hematin and eoin staining. Immediately place the slide into cold, 100%ethanol for two minutes.

After this time, remove the slide and let it dry at room temperature. The h and d stained slide provides better visualization of nuclear textures and should be used by the pathologist to mark the areas of interest. Next, place a room temperature pen frame.

Slide into a frame supporter, adjust the cryostat to 10 microns and create sections. One to four sections can be placed onto each slide, depending on the tissue size. Immediately plunge the slide into cold 100%ethanol.

Remove the slide after two minutes and store in the freezer for later analysis. On the same day of the laser capture microdissection, retrieve the pen frame slides from the freezer working in an RNA free area. Hydrate the slides by gently dipping them into 90%ethanol for two minutes, then 75%ethanol for two minutes.

Once hydrated, stain the prostate tissue by gently dipping the slides into 0.5%toine blue for between five to 30 seconds. Detain the tissue by washing the slides twice in depthy water for 15 seconds, followed by 75%ethanol for 30 seconds to three minutes. Lastly, transfer the pen frame slides to an RNA free dry container on ice.

Before beginning the procedure, place a slide to dry on a slide warmer for 15 minutes. Dry only the slide that will be processed and store the rest on ice until needed. Turn on the LCM components in the following order.

Microscope power, the laser key, the laser switch, and then the computer. Next, launch the laser microdissection software using the software. Prompt the microscope to load the slides and collection tubes.

Click on unload sample holder and load the slide onto the slide holder with the tissue facing down. Insert the slide holder into the appropriate slot in the microscope and click continue. Next, click on unload collection tubes.

Label and load tubes onto the tube holder and insert into the appropriate slot. Secure the caps and add 35 microliters of lysis buffer. Taking care to avoid bubbles.

Once the collection caps are loaded, click okay. Use the software to select the position of the slide in the holder, and then select the collection cap to collect the specimen. Visualize and focus the slide through the computer at 10 x magnification.

Use the software to calibrate the laser by selecting laser. Then calibrate, adjust the power aperture and speed of the laser by selecting laser, and then control. Continue to make adjustments until the laser cuts completely through the tissue.

It is crucial to outline the areas of interest typically done by a board certified pathologist in order to accurately dissect the desire areas of the tissue subjected to LCM Using the eight by 11 h and e pathologist markup as a visual guide. Use the draw shape tool in the software to circumscribe the desired areas of epithelium in the view and avoid collecting any stroma. Click cut to start the laser.

If an area is not completely cut, use the move and cut tool to go over it manually. Continue moving the objective to new views and repeat laser cutting until the slide is completely dissected or the desired amount of tissue has been collected. When complete, carefully remove the tubes from the collection holder and close the caps being mindful of the lysis, buffer and tissue in the caps.

Briefly, centrifuge for 30 seconds to collect the liquid and tissue in the bottom of the tube. Incubate the samples at 42 degrees Celsius for 30 minutes and store at minus 80 degrees Celsius until ready for RNA isolation. These examples show representative toluidine blue stained prostate sections on a pen frame slide before and after laser capturing benign epithelium cell type specific controls.

Confirm the specificity of the laser captured area of the tissue. Benign epithelium and stroma. Do not express the prostate cancer specific gene, A-M-A-C-R.

The stroma did not express the epithelial specific gene and KX 3.1 While attempting this procedure. It's important to remember to work in an RNA free environment to prevent RNA degradation. After watching this video, you should have a good understanding of how to isolate RNA from homogeneous populations of epithelial or storm cells from frozen tissue for numerous downstream analysis techniques.

Don't forget that working with a cryostat can be extremely hazardous in precautions, such as good training and extremely good attention to the blade should always be taken while performing this procedure.