This method can help answer key questions about the enteric nervous system, such as the expression profile of enteric ganglia in the intestines of adult humans. The main advantage of this technique is that it can be used to obtain high yields of RNA from human enteric ganglia while maximally preserving RNA quality. Begin with intestinal tissue cut into 20 centimeter lengths.
First, trim away the adipose and connective tissue. Avoid nicking the serosa, which can lead to structural damage to the myenteric plexus. Next, make a longitudinal incision along the entire length of the trimmed sample, preferably at the site of mesenteric attachment.
Then, dissect away and discard the taeniae coli from the colon so the tissue can flatten out. Now, splay the intestine, mucosa side down, onto a chilled cutting board. Then, cut 1.25 centimeter wide strips along the full length of the tissue.
As they are cut, transfer each piece to chilled tissue storage solution for temporary storage. Next, cut each strip into segments that are two to 1.5 centimeters long. After slicing a strip into segments, blot the segments dry.
It's important that the tissue is very dry so ice crystals don't form on the tissue when it is frozen. Now, arrange the dried pieces of tissue, mucosa side up, on a cold, disposable plastic mold. Then, flatten each piece using curved forceps to remove trapped air bubbles.
The tissues will expand as they are flattened. After flattening the tissue segments, assess their thicknesses prior to freezing. If needed, push the edges of the specimen inward until the intestinal sample approaches its original thickness.
Now, transfer the loaded base mold onto a slurry of dry ice and 2-methylbutane. The tissue should completely freeze within 30 to 60 seconds. Then, immediately dry the bottom of the mold using cold wipes that have been kept on dry ice.
Now, check the sample. Take note of any samples that do not appear flat. Next, wrap the mold in aluminum foil that has been pre-labeled and chilled before use.
Then, cover the foil with a layer of plastic wrap to minimize dehydration. Keep the samples on ice until they can be stored at minus 80 degrees Celsius. After preparing the required materials, pour a three to five millimeter mound of TFM onto a large cryostat specimen holder and immediately transfer the intestinal sample, serosa side up, onto the TFM.
After a few seconds at room temperature, transfer the specimen holder to dry ice and cover it with powderized dry ice. Before slicing, make sure that the TFM is below the plane of the myenteric plexus. Next, mount the specimen holder into the cryostat specimen head and align the specimen with the cryostat blade.
Make the plane of the myenteric plexus parallel to the cutting blade. Now, make eight micron sections through the serosa and longitudinal muscle until reaching the myenteric plexus. To locate the myenteric plexus, mount a sample and stain it with an aqueous dye, such as 1%crystal violet.
To proceed, identify the junction between the longitudinal and circular muscle layers. At the start of sectioning, the serosa will be marked by the presence of connective tissue. Some remaining mesenteric fat may also be present along the serosa.
Then, the sheet of the outer longitudinal muscle layer becomes visible Once the border between the longitudinal and circular muscle layers has been reached, a portion of the enteric ganglia will be observed. In the next several sections, the myenteric plexus will become very evident with large swaths of ganglia being present within a single section. Now, collect serial sections for the laser-capture microdissection.
Chill PEN slides in the cryostat for five to 10 seconds and use them to mount the sections. The best sections will have a lot of neurons and glia. Now, proceed to stain and then dehydrate the sections as described in the text protocol.
In preparation, insert a cartridge of LCM caps into the LCM microscope stage. Then, load the sample slide onto the stage and acquire an overview image of the slide. Identify the desired location for LCM and load a cap onto the slide.
Then, align the infrared laser and adjust the laser's power and duration for a 20 to 30 micron diameter capture spot. Next, outline the desired ganglia to be collected. Make sure to stay within the boundary of the collectible area on the cap, which is displayed as a green circle.
For each of the ganglia outlined by a UV cutting trace, adjust the IR spots such that there is at least one IR spot every 100 to 500 microns to ensure the ganglia will all be collected when the cap is lifted off of the sample. Then, press the IR UV Cut button to proceed with the collection of all the marked ganglia. Once collections are completed, move the cap to a new location and repeat the collection process.
This can be repeated for 60 to 80 minutes. When the LCM cap is loaded with enough ganglia, transfer it to a quality control station. If debris is present on the cap, wipe it away using a fine-tipped paint brush that has been prepared for RNA use.
Use a pipette tip for any difficult to remove debris. Then, secure the cap onto a 0.5 milliliter microfuge tube filled with 230 microliters of RNA lysis buffer. Invert the cap, briefly vortex it, and then incubate the sample at room temperature for 30 minutes.
Later, centrifuge the sample for five minutes at 5, 000 g or faster, and then transfer the microfuge tube to dry ice. The sample is now ready for the RNA extraction. When pulling two caps of enteric ganglia per sample, greater than one nanogram of RNA was obtained, enough for RNA-Seq, and the RNA was of excellent quality.
The RNA integrity of shown sample is 8.3. Samples with integrity numbers of 7.5 and 6.9 were also collected. In total, more than 95%of all prepared samples have been good enough for RNA-Seq.
The quality of the RNA-Seq data sets was assessed using a bioinformatics pipeline. The end bias indicates that the samples were successfully sequenced and only had a modest three prime end bias. A gene biotype plot shows that the sequencing results were largely consistent.
Once mastered, this technique can be used to collect biological replicates of RNA from enteric ganglia of sufficient quality and quantity for RNA-Seq in as little as eight to 12 hours. While attempting this procedure, it is important that the human intestinal samples be frozen as flat as possible. This will ensure that you can collect very large cross-sections of human enteric ganglia that are quickly and easily processed with laser-capture microdissection.
