Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

The animal necropsy procedures depicted here are covered under approved IACUC protocols at the National Animal Disease Center, Ames, IA. All experiments were conducted in accordance with the approved guidelines for animal care and welfare.

1. Pre-planning Before Necropsy

1. Before the necropsy, prepare the following materials for transport to the necropsy suite:
   1. 1.5 or 2 mL microcentrifuge tubes filled with ~ 1.0 mL of RNA preservation solution, in a rack or transport container. Be sure to follow the manufacturer's guidelines for the ratio of the volume of the preservation solution to the volume of the tissue.
   2. Disposable scalpels and clean, autoclaved forceps: one set for dissecting the skin and a different set for collecting the lymph nodes (one for each lymph node, per animal), thus preventing a cross-contamination of the skin and environmental flora with the lymph node tissues.
   3. 3 mm punch biopsy tools, necropsy knives, a sharps container for the used scalpels and biopsy tools, and cutting boards or disposable trays for use in the lymph node dissection.
   4. Use personal protective equipment, including PPE required for work at the appropriate BSL level for the system.
   5. Autoclave reusable metal tools, including the forceps, in metal pans prior to the necropsy, using the utensils/dry goods setting.

2. Identification and Sampling of Lymph Nodes in Cattle and Bison

Figure 3. Lymph nodes of the bovine head and neck. (A) This cartoon image shows selected lymph nodes of the head and neck of Bos taurus. (B) This image shows the parotid lymph node in cross-section (left) as compared to the parotid salivary gland in cross-section (right). Note the difference in textures between the two tissue types. Please click here to view a larger version of this figure.

1. Isolation of prescapular lymph nodes in cattle and bison
   1. Euthanize the animal (cattle or bison of any age, as appropriate for the experiment) based on the local IACUC protocol. Confirm the death by methods specifically described in the institutional IACUC protocol, including the lack of a heartbeat, the lack of respiration, and the absence of a corneal reflex. Move the animal to the necropsy floor.
      Note: There are various methods of humane euthanasia used in the laboratory practice of large animals. However, intravenous administration of pentobarbital sodium and phenytoin sodium solution is the most commonly approved and utilized form. Consult the AVMA Guidelines for the euthanasia of animals.¹⁹
      CAUTION: Follow all relevant biosafety protocols for a necropsy at the host institution, including the guidelines for the safe disposal of sharps in a puncture-proof container, and the decontamination of reusable metal knives and forceps. This protocol generates sharps waste from the use of disposable scalpels. Wear personal protective equipment as dictated by the institutional guidelines, including boots, coveralls, gloves, and eye protection.
   2. Identify 3 participants to work on the animal tissues.
      Note: Here, dissector 1 will be in charge of the large-scale sectioning of the desired animal tissues, dissector 2 will recover the lymph nodes from the different animal sections, and dissector 3 will work at a separate table to isolate sections from each lymph node and transfer them to tubes with a preservative solution.
   3. Begin by placing the animal in lateral recumbency.
   4. Extend the shoulder by moving the front limb back at the level of the knee (carpus); this motion will bring the shoulder back and allow for an easier palpation of the lymph node. Feel for the presence of the lymph node "lump".
      Note: During disease conditions, lymphadenopathy may be present, which makes the identification of lymph nodes easier, but this process can also affect the consistency, composition, and architecture of the lymph node.
   5. 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.
      Note: Do not restrict the size of the incision, even if the lymph node has been located via palpation. A larger incision provides easier access to deeper tissues and makes the excision of the lymph node easier for the dissector.
   6. Using a pair of forceps, retract the skin to reveal the point of the shoulder and neck musculature.
      Note: The prescapular lymph node is often found in subcutaneous fat, especially in overconditioned animals.
   7. Once again, palpate the location of the lymph node (Figure 3A).
      Note: Unlike the subcutaneous fat, the lymph node will hold its shape when palpated. Additionally, lymph nodes are not contiguous with any surrounding tissue, as they are enclosed by a thin fibrous capsule.
   8. After locating the prescapular lymph node, carefully dissect out the subcutaneous fat, using a scalpel and forceps, and isolate the lymph node.
      1. Look for the presence of a thin fibrous capsule, as depicted in Figure 1B, to distinguish between the lymph node and adipose tissue; lymph nodes are not contiguous with any surrounding tissue.
   9. Transfer the lymph node to the lymph node dissection table, using a plastic tray for the tissue transfer, for further sectioning.
      Note: The appearance of these lymph nodes in bison is similar. The associated video depicts the appearance of the prescapular lymph node as dissected out of the right shoulder of an American bison.
2. Isolation of parotid lymph nodes in cattle and bison
   1. Begin by placing the animal in lateral recumbency on the necropsy floor. Locate the temporomandibular joint (TMJ).
      1. If this is difficult to locate, move the mandible slowly and determine the location at which the mandible attaches to the skull; this is the TMJ.
   2. Using a scalpel or necropsy knife, carefully make an incision in the skin. Start the incision dorsal to the joint and extend it ventrally/vertically along the jawline.
   3. Dissect the skin away to reveal the subcutaneous tissue. The parotid salivary gland will be visible first, due to its large size, lobulated surface appearance, and red coloration.
   4. Using forceps and a scalpel, move the parotid salivary gland aside to find the parotid lymph node sitting just cranial and medial to the salivary gland (Figure 3A). Examine the parotid lymph node tissue for texture before further processing.
      Note: Lymph nodes of the face (parotid and submandibular) are associated with salivary glands, which can confound the lymph node isolation. In comparison to lymph nodes, salivary glands are much larger and have a lobulated surface appearance. Moreover, on cut surface, salivary glands are homogenous in nature and do not exhibit the classic cortical and medullary architectural pattern of lymph nodes (Figure 3B).
   5. Excise the gland with a scalpel. Transfer it to the lymph node dissection table, using a plastic tray for the tissue transfer, for further sectioning.
3. Isolation of supramammary lymph nodes in female cattle and bison
   1. Place the animal in lateral recumbency.
   2. Elevate the hind leg to expose the inguinal region and locate the udder.
      Note: In a healthy, non-lactating animal, palpation of these lymph nodes may not be possible. In a lactating animal, these lymph nodes may be palpable at the caudal border of the base of the udder.
   3. Using a scalpel, make an incision just dorsal to the udder, running along the upper leg, to expose the supramammary glands.
   4. Dissect the subcutaneous tissue and find the supramammary lymph node embedded in a thin layer of subcutaneous fat. If not visually distinguishable, palpate the exposed tissue for the presence of a firm structure within the subcutaneous fat.
   5. Using forceps and a scalpel, carefully dissect out the lymph node from the fat. Transfer it to the lymph node dissection table, using a plastic tray for the tissue transfer, for further sectioning.

3. Sectioning and Storage of Lymph Nodes

1. Pass the isolated lymph nodes from dissector 2 to dissector 3 at a dissection table adjacent to the main necropsy space. Place each lymph node on a fresh section of a cutting board. Pre-label the cutting boards in sections by lymph node, to facilitate the receipt of multiple lymph nodes in succession. For infectious samples, use disposable trays.
2. Using forceps and a disposable scalpel or necropsy knife, remove a section of the lymph node. Using a sharp knife, make a sagittal cut to open up the lymph node.
3. Examine the interior of the lymph node, especially in specimens from infected animals, looking for asymmetry, lesions, color differences, etc. Record the observations.
4. (Option 1) Small tissue sections
   1. Use disposable scalpels to excise pieces of each lymph node that are less than 5 mm in thickness and transfer each piece to a tube with an RNA preservation solution with clean forceps.
5. (Option 2) Lymph node biopsy or sectioning methodologies
   Note: Biopsies or sectioning methodologies, when applied to parallel lymph node sections across different nodes and animals, provide consistency of sampled tissue for a bulk RNA analysis.
   1. Wedge sectioning method
      1. After examining the sagittal section to capture cells across the profile of the lymph node, excise a pie-shaped wedge from the lymph node, from the center to the outer capsule. For a node 2 cm in width (standard size; see Table 1), a wedge cut to the center that is 4 mm in outer arc length and 5 mm in thickness will have a wet weight of ~ 100 mg.
      2. Using the scalpel, cut the wedge into small pieces, no thicker than 5 mm, and approximately 50 – 100 mg each in wet tissue weight.
         Note: For consistency, process all wedge pieces from a single lymph node either in one batch or in separate tubes followed by RNA pooling to provide a representative RNA profile from the lymph node of interest.
      3. Place the tissue pieces with forceps into tubes containing at least 10 volumes of RNA preservation solution, as compared to the volume of the tissue.
   2. Punch biopsy method
      1. Select a punch biopsy tool if the goal is to collect specific sections, such as specific follicles from the node. Select a tool consistent with the size of the sample desired for collection. Punch biopsy tools are available in a range of sizes, from 1 to 8 mm in diameter.
      2. Visually locate the regions of interest to sample in the lymph node.
      3. Use the punch biopsy tool to excise the sections of interest, pressing and turning the tool into the tissue to create the core. Transfer the tissue piece with forceps to a tube containing at least 10 volumes of RNA preservation solution. If thicker than 5 mm, use forceps and a scalpel to further divide the tissue into smaller pieces.
6. Once the samples have been transferred to preservation solution (e.g., RNALater), transport them back to the laboratory at room temperature.
7. Store the samples at 4 °C overnight to allow for tissue penetration.
8. The next day, pour off the excess RNA preservative solution and store the tissue samples at -80 °C. Remove as much preservative as possible before freezing the samples. Use a P1000 and/or P200 micropipette tip for an efficient RNA preservative solution removal.
   CAUTION: Discard the decanted preservation solution appropriately, based on the infective properties of the pathogen utilized as well as the chemical and environmental hazards of the preservative solution as specified on the Safety Data Sheet.
   Note: The guidelines provided here are provided for the RNA preservation solution described in the Table of Materials. Consult the manufacturer's guidelines if using alternative preservation solutions.

4. Processing from RNA Lymph Nodes

CAUTION: Wear a lab coat, gloves, and proper eye protection for the processing steps.

Note: The phenol-based reagent used here is described in the Table of Materials (and the protocol is based on the manufacturer's guidelines)²⁰. The use of alternative phenol-based reagents may necessitate a modification of the procedure, based on the manufacturer's recommendations for the specific product purchased.

1. Prior to accessing the samples, pre-aliquot the chilled (4 °C) phenol-based reagent to homogenization tubes (1.5 mL/tube), using a pipette.
   CAUTION: Phenol is toxic, corrosive, and a health hazard. Chloroform is toxic and a health hazard. Complete processing steps 4.1 – 4.9 in a chemical fume hood, and wear gloves to prevent any contact with the chemicals. In the United States, chloroform is a hazardous waste; dispose of tubes containing hazardous waste according to local and institutional guidelines. Process tissue samples from animals infected with pathogens under the corresponding BSL-2, BSL-3, or BSL-4 guidelines.
2. Once a sample can be loosened from the microcentrifuge tubes with clean forceps, immediately transfer approximately 50 – 100 mg of the preserved tissue to the homogenization tube containing the phenol-based reagent.
   1. Limit the thawing prior to the addition to the phenol-based reagent. Use storage on dry ice when multiple samples are removed from the freezer for processing.
3. Tightly cap the tube, place it on the homogenizer, and homogenize the sample with a rotor-stator tissue homogenizer in the phenol-based reagent. Complete the processing at room temperature. When complete, check for a cloudy appearance to ensure the sample was successfully homogenized; the tissue should be dispersed into a cloudy suspension.
   Note: A 2 min RNA extraction setting is used (the pre-set RNA_02_1 setting for the homogenizer described in the Table of Materials; designed for frozen tissues). The rotor-stator specified in this protocol (see the Table of Materials) is large-toothed and adapted for the homogenization of a range of tissue types, including fibrous material. The RNA_02_01 setting is designed specifically for frozen (as opposed to fresh) tissues. As lymph nodes have fibrous connective tissue components, a similarly optimized rotor-stator homogenizer is recommended to achieve an effective dissociation. See the National Institute of Environmental Health Sciences²¹ for additional information about homogenization recommendations.
   1. If large chunks of tissue remain after the homogenization, repeat the homogenization procedure. In order to efficiently recover the RNA, the tissue must be well dissociated.
   2. For more fibrous lymph node pieces, use forceps and scissors to pre-chop the lymph node piece into several smaller pieces prior to the transfer to the homogenization tube. As described in the introduction, lymph node tissues from older animals may be more fibrous.
      Note: If a dual analysis of the host and pathogen RNA is desired, additional treatment (such as bead homogenization) may be necessary for the recovery of bacterial RNA, especially if Gram-positive pathogens are present in the tissues.
4. Immediately remove the homogenized sample from the tubes and transfer it to RNase-free microcentrifuge tubes (2.0 mL in size) with a P1000 micropipette.
5. Centrifuge the sample for 5 min at 12,000 x g at 4 °C to remove any sample debris. Using a P1000 micropipette tip, transfer the supernatant to a fresh microcentrifuge tube, leaving the pellet behind.
6. Incubate the supernatant for 5 min at room temperature. Split each sample between two 1.5 microcentrifuge tubes.
7. Add 0.2 mL of chloroform per 1 mL of the phenol-based reagent (i.e., 0.3 mL for a 1.5 mL sample); invert it by hand for 1 – 2 min to mix the phases, but do not vortex the sample. Incubate it for 2 min at room temperature.
8. Centrifuge it for 15 min at 12,000 x g at 4 °C.
9. Carefully remove the upper, clear aqueous phase with a micropipette tip (P1000 or P200) which will form the upper layer, without disrupting the interphase or the red phenol-chloroform layer. Transfer it to a new microcentrifuge tube.
10. Mix it with an equal volume of 100% ethanol; mix it thoroughly by inverting the tube 4 – 5 times. The tube will often appear cloudy.
11. With a micropipette tip, transfer the liquid to a silica-based commercial spin column, to further purify and elute the RNA, following the manufacturer's instructions²². Elute the RNA generated from ~ 50 mg of tissue into 50 – 100 µl of RNase-free water.
    Note: While the phenol-based reagent extraction removes DNA in the organic phase, for the downstream usage of RNA in qRT-PCR and/or RNA-sequencing applications, an additional treatment of RNA samples with DNase is recommended.
12. Aliquot the eluted RNA to separate tubes for use in a quantification and quality assessment, to reduce any freeze-thawing of the main RNA sample. Transfer the tubes to -80 °C for storage.
13. In the case of lymph node samples derived from animals infected with zoonotic pathogens, especially at the BSL-3 containment level, validate the resulting RNA sample for the absence of pathogens if the sample will be moved to a lower biosafety level containment area for quality analysis, RT-PCR, and/or RNA-sequencing library preparation.
    Note: It is critical to consider local regulations, and in the case of the CDC (Centers for Disease Control and Prevention) inactivation guidelines for work with select agents, it is necessary to validate all inactivation procedures at the researcher's local site.
    1. Remove 1/10^th volume of each eluted RNA sample from step 4.12 (i.e., 5 µl from 50 µl of the total RNA). Using a sterile technique, mix the sample with 100 µl of bacterial growth broth in a sterile 1.5 mL microcentrifuge tube. Using a sterile plastic spreader, spread the RNA-broth mixture across the surface of an agar plate.
       Note: Select a broth type and agar plate consistent with the growth of the pathogen with which the animals were challenged.
    2. Incubate the agar plate under conditions specific to the growth of the pathogen with which the animals were challenged. Check for the absence of recovered bacteria before removing the samples from the BSL-3 containment.
       Note: The resulting eluted RNA, after quantification and tests of integrity, is suitable for downstream applications, including an RNA-sequencing and RT-PCR analysis.
14. Assess the RNA recovery and purity using a spectrophotometer. To assess the RNA quality, load 1 – 2 µl of the eluted RNA sample to the spectrophotometer for an analysis. Record a spectrum of the sample in the ultraviolet (UV) range.
    1. From the UV spectrum, record the A₂₆₀/A₂₈₀ ratio, the A₂₆₀/A₂₃₀ ratio, and the A₂₆₀ value for the sample (A = Absorbance).
    2. As single-stranded RNA at a concentration of 40 µg/mL has an absorbance of 1.0 at 260 nm, calculate the RNA concentration (µg/mL) by multiplying the A₂₆₀ x 40 for the purified samples.
15. As a quick method of assessment of RNA integrity, to exclude any degraded samples from further analysis, mix 2 µl of each RNA sample with 4 µl of 1.5x formamide loading dye [95% deionized formamide, 0.025% (w/v) bromophenol blue, 0.025% (w/v) xylene cyanol, 5 mM EDTA (pH 8.0), 0.025% (w/v) SDS] in 1.5 mL microcentrifuge tubes^23,24.
16. Heat the tubes at 70 °C for 1 min, and then transfer the tubes to ice for 1 min.
17. Load the samples to a 1% agarose gel prepared in 1x TBE (for a full description of a native agarose gel electrophoresis for RNA, see Rio, Ares Jr., Hannon, and Nilsen)²⁴. Separate the samples by standard gel electrophoresis methods and confirm the presence of 28S and 18S ribosomal RNA bands.
18. Follow up the gel electrophoresis with quantitative analysis methods for the RNA integrity determination [e.g., a Bioanalyzer and RIN (RNA Integrity Number) analysis] as described in the Representative Results and in Mueller, O. et al.²⁵ and Schroeder, A. et al.²⁶.

5. Alternative Extraction Method from Formalin-fixed, Paraffin-embedded (FFPE) Tissues

Note: Although FFPE tissue preservation does not represent the most robust method of nucleic acid preservation, the protocol presented below can be a way to study some transcriptional changes when other preserved tissues are unavailable.

1. Prepare formalin reagents for the tissue preservation by dispensing formalin into plastic containers. Tissues should be preserved in formalin with a ratio of 20:1 formalin:tissue volume/weight, respectively.
   CAUTION: Formalin is a known human carcinogen and although not acutely toxic, chronic exposure (typically through inhaled fumes) can represent a significant health hazard. Proper ventilation and PPE (i.e., the use of nitrile gloves, to be changed within 15 min after the initial exposure) are required to minimize the health risks. Refer to the OSHA Formaldehyde Standard (29 CFR 1910.1048) for additional information on exposure monitoring and risk assessment.
   Note: Using too little formalin can impact the ability of the tissue to become fully preserved when submerged in preservative.
2. After the isolation of the tissue of interest, carefully trim the tissue to less than 5 mm in thickness.
   1. Using forceps, carefully submerge the trimmed tissue into formalin to minimize any splashing.
      Note: The tissues should be completely submerged in formalin to allow for a complete fixation. It is critical that all surfaces of the tissue are completely covered with formalin.
3. Once the tissues have been submerged in formalin, allow for the tissue fixation to occur for at least 24 h at room temperature.
   Note: Formalin penetrates tissues slowly, at a rate of 1 mm per hour from all surfaces^27,28. Therefore, keeping the thickness of the tissue sections to less than 5 mm results in a rapid fixation of the entire tissue.
4. After the fixation, transfer the tissues to an appropriate reagent for the paraffin embedding.
   Note: For example, the tissues examined in this manuscript were transferred to 70% ethanol prior to the embedding.
5. Embed the tissues into paraffin.²⁹
6. Section the tissue to 10 – 80 µm in thickness (use 10-20 µm if miRNA is desired to be extracted)²⁹.
7. Using scalpel and forceps, remove a 40 mg tissue piece from each sample to be processed and place it into a sterile, nuclease-free microfuge tube.
8. Utilize a commercial kit designed for deparaffinization and nucleic acid recovery of FFPE tissue samples to extract RNA from the preserved samples.
   Note: The kit referenced in the Table of Materials utilizes xylene and ethanol washes to remove paraffin, a protease treatment step, and a glass-fiber filter to purify RNA.