Coleta e processamento de linfonodos de animais de grandes porte para a análise do RNA: preparando-se para estudos de linfonodo Transcriptomic de espécies de animais grandes

Hi, my name is Dr.Robert Schaut and I'm a scientist at the National Animal Disease Center in Ames, Iowa. In this JoVE video, we will be sharing with you information about the collection and processing of lymph nodes from large animals for use in downstream RNA experiments. For example, these experiments might include quantitative PCR on reverse transcribed products, or RNA sequencing studies of transcriptomes.

Lymph nodes are useful samples for the assessment of host immune responses but can also provide a source of pathogen RNA for study, such as the case of intra-cellular pathogens present in immune cells inside lymph nodes. Samples from large animals, including both livestock and wildlife species, are of interest in the study of zoonotic diseases. Here, at the National Animal Disease Center, we study infections of livestock with species like brucella abortus, which causes brucellosis in cattle and bison, and E.Coli O157:H7 as shed from the intestinal tracks of cattle.

We will also recover tissue samples from wildlife species, like deer and elk. A range of lymph nodes can be collected from animals infected with different diseases. Studies of tissues in these natural host species are generally more relevant to the natural progression of disease than mouse models.

However the process of tissue harvesting from large species is necessarily more complicated than from mice, including the longer delay between euthanasia and tissue collection, and a larger size of lymph nodes. Here, we'll present a protocol for the isolation of large animal lymph nodes for RNA study that will be informative for molecular biologists involved in the transcriptomic studies of large animal infections, as well as veterinary researchers commencing transcriptomic studies. In this video, we will be demonstrating procedures on lymph nodes for cattle.

Before necropsy, we recommend assembling the following supplies specific for tissue isolation procedure. Microfuge tubes filled with about 1ml of RNA tissue preservative, like RNAlater, disposable scalpels, clean and autoclave forceps, biopsy needles, a sharps container for used scalpels and needles, and either a cutting board or disposable tray, depending on the level of infectivity of the materials involved. In this case, we will demonstrate key elements of the procedure by using lymph nodes that we generally collect from cattle.

They're infected with bacteria that cause brucellosis. These are the prescapular lymph nodes, and the parotid lymph node. When conducting a large animal necropsy for RNA recovery, it's useful to have at least three participants working on animal tissues.

Dissector one will be in charge of large animal sectioning of different pieces of the animal, dissector two will recover lymph nodes from different animal sections at the main necropsy table, and dissector three will work at a separate table to isolate sections from each lymph node, and transfer them to tubes with preservative solution. We will start with the identification and removal of the prescapular lymph node. Ideally, palpation of a lymph node, prior to dissection, will help identify the location of the lymph node.

While not always possible to detect, the prescapular lymph node is typically palpable in a healthy animal. Begin by placing the animal in lateral recumbency. Extend the shoulder by moving the front limb back at the level of the knee.

This motion will bring the shoulder back, and allow for easy palpation of the lymph node. Feel for the presence of the lymph node as shown. Once located, and with the shoulder still extended, use a scalpel or necropsy knife to make a skin incision along the outline of the shoulder.

Retract the skin to reveal the point of the shoulder and neck musculature. A section of subcutaneous fat is sometimes observed, especially in over-conditioned animals. The prescapular lymph node is often found with sections of subcutaneous fat.

Here you can see the three layers of the skin, muscle, and subcutaneous fat. Once again, palpate the location of a lymph node. Unlike the subcutaneous fat, the lymph node will hold its shape when palpated.

After locating the prescapular lymph node, carefully dissect out the subcutaneous fat, and dissect out the lymph node. The appearance of these lymph nodes in bison, which we used in brucellosis studies due to the susceptibility of bison in the Greater Yellowstone area to brucella, is similar. Here, we demonstrate the appearance of the prescapular lymph node, as dissected out of the right shoulder of an American bison.

Next we will move to the isolation of the parotid lymph node. With the animal in lateral recumbency, locate the temporomandibular joint. Using a scalpel or necropsy knife, carefully make an incision on the skin.

Start dorsal to the joint, and extend vertically along the jaw line. Dissect the skin away to reveal the subcutaneous tissue. The parotid salivary gland will be visible, first due to it's large size, lobulated surface appearance, and red coloration.

Using forceps and scalpel, move the parotid salivary gland aside to find the parotid lymph node sitting just cranial and medial to the salivary gland. When isolating the parotid lymph node, it's critical to separate lymph node tissue from the tissue of the parotid salivary gland. The tissue types look distinct, including within the tissue cross-section.

This can be observed here, with the parotid lymph node on the left, and the parotid salivary gland on the right. Be sure to carefully examine the parotid lymph node tissue for texture before further processing. In some cases, it may be necessary to excise the parotid salivary gland in order to locate the parotid lymph node.

Here is an example of a parotid gland excised from a steer, in which the lymph node was embedded within the salivary gland and not visible before gland removal. Additional information about identification of selected lymph nodes is provided in the main manuscript. Once the lymph nodes have been collected, you're ready to excise sections for preservation.

Use a scalpel or necropsy knife, remove a section of the lymph node, and make a sagittal cut to open up the lymph node. Examine the interior of the lymph node, especially in specimens from infected animals, looking for asymmetry, lesions, color differences, etc. In this bovine prescapular lymph node, multiple features are visible to the naked eye.

These include follicles which are home to B lymphocytes, and deep cortical units, which are home to T cells. These reasons appear darker than the medullary sinuses, which are lighter. Here, another example of an uninfected lymph node is depicted.

This lymph node is from a younger bison. Note the follicles are less distinct in this example. In order to section tissues for processing, one option is to collect small tissues pieces from each lymph node with a scalpel for subsequent RNA recovery.

However one major challenge with comparing expression profiles between samples is the large size of large animal lymph nodes, as compared to a mouse lymph node which can be easily extracted whole for RNA extraction. As a result, small tissue sections will not evenly capture the profile of cells across a lymph node. Instead to capture cells across the profile of a lymph node, excise a pie-shaped wedge from the lymph node, cut into small pieces no thicker than 5mm, and placed, with forceps, into tubes containing RNAlater.

Alternatively utilize biopsy methodologies to excise tissue sections from each lymph node. Select the punch biopsy tool if you would like to collect specific sections, such as specific follicles from the node, and then transfer, with forceps, to RNAlater. Once samples have been transferred to RNAlater, transfer them back to the lab and store at 4 degrees celsius overnight to allow for tissue penetration.

Afterwards, poor off excess RNAlater and store tissue samples at 80 degrees celsius. A plus of using RNA preservative solution is that liquid nitrogen is not required at the necropsy, nor upon collection of samples from the field. Prior to accessing samples, pre-aliquot Trizol reagent to tubes for homogenization.

After samples are removed from the freezer, transfer them immediately to Trizol reagent for subsequent processing, based on manufacturer's recommendations. Here, a piece of 50-100mg of tissue are transferred to Trizol reagent. Limit thawing prior to Trizol addition as much as possible.

Storage on dry-ice can be used when multiple samples are removed from the freezer for processing. Place tubes on the homogenizer, and homogenize in Trizol reagent. Here, a two minute RNA extraction setting is used.

When complete, check for a cloudy appearance. Repeat if necessary. Follow the manufacturer's protocol to generate aqueous and organic layers for each sample.

Remove the upper clear aqueous phase with a pipette tip. Transfer to a new microcentrifuge tube. Next, mix with an equal volume of 100%ethanol.

Mix thoroughly. The tube will often appear cloudy. With a micro-pipette tip, transfer the liquid to a silica-based commercial spin column to further purify and then elute your RNA.

To assess RNA quality, load one or two l of the eluted RNA sample to a spectrophotometer for analysis. After completion of the procedure, we observe absorbance ratios of approximately two for the 260-280 ratio and the 260-230 ratio, indicating a high purity with minimal chemical carryover. Phenol contamination would be observable from a decrease in the 260-230nm absorbance ratio.

During the necropsy procedure in the case of animals like bison, we observed a time period of at least 20 minutes between euthanasia and a transfer of the first tissue sample to preservative. In contrast, mouse lymph nodes can be removed and preserved after euthanasia in a matter of minutes. Therefore we were interested in characterizing the degradation profile of lymph nodes over time.

In the main manuscript we also compiled a summary of RNA stability profiles in a range of other tissue types that have been tested in other publications. In representative experiments, conducted with bison lymph nodes, RNA stability was relatively consistent over the course of an hour after euthanasia. Here, we depict a gel image of total RNA purified from pieces of supramammary lymph node tissue from an American bison.

This finding is consistent with previous data for other tissue types compiled from the literature. This suggests that samples of acceptable quality for downstream analysis of gene expression can be collected even from more challenging species like bison and elk. However it is important to appreciate that longer times between death and sample preservation can result in changes in RNA expression profiles, including increase in degradation from three prime end of mRNA transcripts.

Therefore it is still important to utilize a rapid, streamlined workflow for tissue recovery in order to reduce further delay once the animal is available for necropsy, in order to achieve the most biologically valid RNA profiles. In addition, we note that care must be taken throughout the procedure to avoid introduction of RNA contaminants into the samples throughout the process, beyond those that are already present within the tissues. This includes the use of gloves as part of the other required personal protective equipment.

We also aliquot the final RNA product into multiple tubes in order to limit freestalling of RNA samples as much as possible. For example, separate aliquots can be stored for use in quality control tests. In summary, in this video we have provided important information for the use in identification, collection, representative sampling, and processing of lymph nodes for downstream gene expression analysis.

While our representative video has been demonstrated with cattle and bison examples, we have also worked with lymph nodes from other large species, including goats and elk. We hope you'll find this video useful in your work, whether you are a veterinary scientist considering the use of transcriptomic methods in your work, or a molecular biologist interested into venturing into the field of large animal science.