We are greatly appreciative for the assistance from the Cattle Fever tick Laboratory in Edinburg, Texas. We would like to thank Michael Moses, Jason Tidwell, James Hellums, Cesario Agado, and Homer Vasquez. We would also like to acknowledge the assistance of Sarah Sharpton, Elizabeth Lohstroh, Amy Filip, Kelsey Johnson, Kelli Kochcan, Andrew Hillhouse, Charluz Arocho Rosario, and Stephanie Guzman Valencia throughout the project. We would like to thank the Texas A&#38;M Aggie Women in Entomology (AWE) Writing Group for their help and advice during the writing of this manuscript. The following reagents were provided by Centers for Disease Control and Prevention for distribution by BEI Resources, NIAID, NIH: Ixodes scapularis Adult (Live), NR-42510. Female I. scapularis ticks were also received from the Tick Rearing Facility at Oklahoma State University. This project was funded by Texas A&#38;M University T3: triads for transformation grant and the cooperative agreement #58-3094-1-003 by the USDA-ARS to AOC.

The authors declare no conflict of interest.

The current protocol provides a detailed methodology for extracting miRNA from salivary glands and EVs. However, there are important considerations, all of which are detailed in the notes for each section of this protocol. The capsule and mesh netting must be secured during tick feeding to prevent ticks from escaping. The capsule preparation and placement are described in Koga et al.40. Several replicates of the tick dissections need to be done if an unsuitable sample is discarded. Additionally, s...

Ticks are ectoparasites that vector many pathogens to wildlife, livestock, humans, and their pets1,2. Tick feeding results in significant economic loss by causing damage to hide, reducing weight and milk production due to severe anemia, and the transmission of potentially deadly disease-causing pathogens1,3,4,5. Current control practices for managing tick populations are focused on the use of acaricides. Nevertheless, the continuous emergence of acaricide resistance in ticks parasitizing livestock5,6, the increased incidence of tick bites7, and pathogen transmission within residential areas8,9, have led to a need for unique tick control alternatives.
The tick salivary glands are essential organs that ensure a tick&#39;s biological success. They are formed by different acinus types (I, II, III, and IV) with various physiological functions. The salivary glands are responsible for osmoregulation, both off and on the host, by returning water excess and iron content to the host via salivation2,10. Type I acini are also involved in the uptake of water from the atmosphere by the secretion of hygroscopic saliva10,11. Salivary effector proteins, such as cement and cystatins, are produced within secretory cells in type II and III acini10,12. Type I acini do not affect tick feeding, indicating that the bloodmeal intake does not trigger morphological and physiological changes in these acini type13,14. On the other hand, Acini type II and III are activated during feeding and present very little activity pre-attachment. Thus, feeding is necessary to trigger the enlargement of the secretory cells within type II acini and the production of bioactive compounds. Type III acini are reduced in size during feeding due to the secretion within the secretory granules12.
The salivary glands are also the site of pathogen infection in the tick and route of transmission. During feeding, ticks secrete several compounds with pharmaceutical effects that are needed for successful completion of the bloodmeal10,15,16. These compounds have anti-inflammatory, immunosuppressive, and vasodilatory properties10,15,17. Recent studies have shown that extracellular vesicles (EVs) derived from tick salivary glands harbor several of these compounds, inducing anti-inflammatory and immuno-modulatory effects18,19,20. &#34;Extracellular vesicles&#34; is an umbrella term used to describe vesicles classified as exosomes and microvesicles based on their size and biogenesis. Overall, EVs are lipid blebs with bilayer membranes that are ~40 nm-1 &#181;m in size21; generally, exosomes are described as being 40-150 nm in size, whereas microvesicles are between 150 nm-1 &#181;m in size21,22,23. However, the size is not indicative of the EVs biogenesis pathway22.
The biogenesis of exosomes starts with the sequential invagination of the plasma membrane. This invagination leads to the formation of multivesicular bodies and finally results in the deformation of the vesicular membrane by the action of ESCRT complexes or sphingomyelinases (sMases)24,25. The exosomes can either be lysed within the lysosomes to maintain cellular homeostasis or exit via vesicular fusion to the plasma membrane to deliver cellular constituents to the recipient cells21,24. On the other hand, microvesicles are formed by the action of flopasses and flipasses, changing the conformation of lipids in the plasma membrane26. EVs are essential for cell-to-cell communication, serving as a transport system for intracellular cargo, such as lipids, proteins, nucleic acids, and microRNAs (miRNAs)21,27,28. Once transported, these vesicles deliver their cargo into the cytoplasm of the recipient cells, generating phenotypic changes in the receiving cell22,29. Due to the importance of extracellular vesicles in tick feeding and the manipulation of host immune and wound healing responses18,20, the cargo within extracellular vesicles presents potential targets for the development of anti-tick therapeutics and a unique mechanism to disrupt tick feeding. This includes miRNAs within tick salivary glands and salivary gland-derived extracellular vesicles.
miRNAs are short non-coding sequences, ~18-22 nucleotide (nt) in length, that can post-transcriptionally regulate, degrade, or silence mRNA sequences30,31. During transcription, the pri-miRNAs are cleaved by Dicer (RNA polymerase III) to form a distinctive hairpin-like structure, becoming a pre-miRNA. The pre-miRNA is cut once again by Drosha (RNA polymerase III) to form a mature miRNA duplex. The mature sequence becomes integrated into the RNA-induced silencing complex (RISC) complementary to the mRNA sequence, causing translation repression or mRNA degradation28,30,32. During host feeding, miRNAs within the tick saliva can modulate host gene expression to suppress immune responses and enhance pathogen transmission33,34,35,36,37. Although extensive studies on EVs and miRNAs exist, their roles during feeding at the tick-host interface are still poorly understood. Optimizing protocols that can easily result in the isolation and purification of high-quality miRNAs is crucial for advancing our knowledge on these topics.
Multiple options can be utilized to isolate EVs, such as ultracentrifugation, exosome precipitation, polymer precipitation, immunoaffinity chromatography, and size-based exclusion techniques38. However, these techniques cannot distinguish between exosomes or microvesicles. Thus, as mentioned previously, EV is used as an umbrella term when isolating EVs from different samples. The vesicles isolated in the experiments described herein represent a mixture of vesicles derived from different biogenesis pathways. Further purification of a specific population of extracellular vesicles can be achieved by immunoprecipitation using beads coated with antibodies against markers (i.e., exosomal markers, tumor markers) unique to the vesicle population of interest39,40. miRNAs can also be extracted via different commercially available isolation kits7,41,42.
The objective of this project was to develop a protocol that combines commonly applied methods to isolate EVs and extract miRNA from both EVs and fed-tick salivary glands. Because the secretion of bioactive compounds is activated by feeding12, ticks should be allowed to feed to identify miRNAs that may be important for manipulating host immune and wound healing responses. The present protocol requires a small number of ticks (20 ticks) to isolate EVs and their respective miRNAs, compared to other previously described studies that required 2000 ticks43. Further, it avoids the contamination of salivary secretions with pilocarpine44, which could influence experiments studying the effect of EVs and their miRNAs on host cells.

<table><tbody><tr><td>0.22 &amp;micro;m syringe filter</td><td>GenClone</td><td>25-240</td><td></td></tr><tr><td>1 &amp;micro;m nylon syringe filter</td><td>Tisch Scientific</td><td>283129028</td><td></td></tr><tr><td>1 inch black adhesive</td><td>Amazon</td><td>B00FQ937NM</td><td>Capsule</td></tr><tr><td>10 mL needeless syringe</td><td>Exelint</td><td>26265</td><td></td></tr><tr><td>3&#039; and 5&#039; Adapters</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-Seq Kit</td></tr><tr><td>4 mm vannas scissors</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td><td>Sigma-Aldrich</td><td>1.1523</td><td></td></tr><tr><td>70Ti rotor</td><td>Beckman Coulter</td><td>337922</td><td></td></tr><tr><td>Amphotericin</td><td>Corning</td><td>30-003-CF</td><td></td></tr><tr><td>Beads</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-Seq Kit</td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td>G2939BA</td><td></td></tr><tr><td>Bioanalyzer kit</td><td>Agilent</td><td>5067-1513</td><td></td></tr><tr><td>Centrifuge 5425</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Macron</td><td>UN1888</td><td></td></tr><tr><td>Cyverse Discovery Enviornment</td><td></td><td></td><td>https://cyverse.org/discovery-environment</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>Double-sideded carpet tape</td><td>amazon</td><td>&amp;lrm;286373</td><td></td></tr><tr><td>Falcon Tubes, 50 mL</td><td>VWR</td><td>21008-940</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>FBS-02-0050</td><td></td></tr><tr><td>fine forceps</td><td>Excelta</td><td>5-S-SE</td><td></td></tr><tr><td>Foamies, 2 mm</td><td>Amazon</td><td>B004M5QGBQ</td><td>Capsule</td></tr><tr><td>Isoflurane</td><td>Phoenix Pharmaceuticals manfactured</td><td>193.33165.3</td><td></td></tr><tr><td>Ixodes scaplaris</td><td>CDC, Oklahoma State University</td><td></td><td></td></tr><tr><td>L15C300 medium</td><td>In-lab</td><td></td><td></td></tr><tr><td>lipoprotein-cholesterol concentrate</td><td>MPI</td><td>02191476-CF</td><td></td></tr><tr><td>Microscope slide</td><td>VWR</td><td>10118-596</td><td></td></tr><tr><td>miRDeep2</td><td></td><td></td><td>https://github.com/rajewsky-lab/mirdeep2</td></tr><tr><td>M-MuLV Reverse Transcriptase</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-Seq Kit</td></tr><tr><td>molecular grade ethanol</td><td>Fischer Bioreagents</td><td>UN1170</td><td></td></tr><tr><td>multi-well 24 well tissue culture treated plate</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Nanopaticle Tracking Analyzer machine</td><td>Malvern Panalytical</td><td></td><td></td></tr><tr><td>Nanosep with 300K Omega filter</td><td>Pall Corporation</td><td>OD3003C33</td><td></td></tr><tr><td>NEXTFLEX Small RNA-Seq Kit v3</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>NextSeq 500/550 High Output Kit (75 cycles)</td><td>Illumina</td><td>20024906</td><td></td></tr><tr><td>Optima XPN 90 Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Penicillin</td><td>Thermofischer Scientific</td><td>ICN19453780</td><td></td></tr><tr><td>Pippettes</td><td>Ependorff</td><td></td><td></td></tr><tr><td>polycarbonate centrifuge bottle</td><td>Beckman Coulter</td><td>355618</td><td></td></tr><tr><td>Qiagen miRNeasy kit</td><td>Qiagen</td><td>217084</td><td></td></tr><tr><td>QIAzol lysis reagent</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Qubit</td><td>Thermofisher</td><td>Q32880</td><td></td></tr><tr><td>Qubit kit</td><td>Thermofisher</td><td>Q10212</td><td></td></tr><tr><td>Rabbits</td><td>Charles River</td><td></td><td></td></tr><tr><td>Reverse Universal Primer</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-Seq Kit</td></tr><tr><td>Rhipicephalus microplus</td><td>Cattle Fever Tick Research Labratoty</td><td></td><td></td></tr><tr><td>Rifampicin</td><td>Fischer Bioreagents</td><td>215544</td><td></td></tr><tr><td>RNAlater</td><td>Invitrogen</td><td>833280</td><td></td></tr><tr><td>RNAse free tubes</td><td>VWR</td><td>87003294</td><td></td></tr><tr><td>RNAse inhibitor</td><td>Thermo Fischer</td><td>11111729</td><td></td></tr><tr><td>RNAse/DNAse free water</td><td>Qiagen</td><td>217084</td><td></td></tr><tr><td>RNeasy Minelute spin column</td><td>Qiagen</td><td>217084</td><td>Qiagen miRNeasy kit</td></tr><tr><td>RPE Buffer</td><td>Qiagen</td><td>217084</td><td>Qiagen miRNeasy kit</td></tr><tr><td>RT Buffer</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-seq kit</td></tr><tr><td>RT Forward Primer</td><td>Illumina</td><td>20024906</td><td>NEXTFLEX Small RNA-seq kit</td></tr><tr><td>RTE Buffer</td><td>Qiagen</td><td>217084</td><td>Qiagen miRNeasy kit</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014-25G</td><td></td></tr><tr><td>Sorvall ST16</td><td>Thermo Fischer</td><td>75004380</td><td></td></tr><tr><td>Sterilized Gauze sponges</td><td>Covidien</td><td>2187</td><td></td></tr><tr><td>Sterilized PBS</td><td>Sigma</td><td>RNBK0694</td><td></td></tr><tr><td>streptomycin</td><td>thermofischer Scientific</td><td>15240062</td><td></td></tr><tr><td>TapeStation</td><td>Aligent</td><td>G2991BA</td><td></td></tr><tr><td>Tear Mender Instant Fabric and Leather Adhesive</td><td>Amazon</td><td>7.42836E+11</td><td>Capsule</td></tr><tr><td>Tissue Adhesive</td><td>3M VetBond</td><td></td><td></td></tr><tr><td>Triple Antibiotics</td><td>dechra</td><td>17033-122-75</td><td></td></tr><tr><td>Tryptose phosphate broth</td><td>BD</td><td>BD 260300</td><td></td></tr></tbody></table>

isolation of micrornas from tick ex vivo salivary gland cultures and extracellular vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

The present protocol provides a detailed methodology to extract miRNAs from salivary glands and EVs. According to the results, this protocol is effective for the isolation of miRNA from adults of two different tick species, I. scapularis and R. microplus, and can potentially be used in other tick species as well. The EVs concentration (particles/mL) was measured via NTA. For R. microplus, each gender and life stage contained three biological replicates measured in three technical repli...

Watch this Scientific Journal Video about Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles at JoVE.com

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

isolation-micrornas-from-tick-ex-vivo-salivary-gland-cultures

Research

JoVE Journal

Biology

2.5K Views.  Texas A&M University. Dit protocol stelt ons in staat om een verminderd aantal teken te gebruiken, wat de kosten van kolonieonderhoud vermindert en het aantal werveldieren vermindert dat nodig is voor het voeren van teken. Dit protocol vereist ongeveer 20 teken. Het belangrijkste voordeel naast het gebruik van een kleine steekproefgrootte is echter dat dit protocol kan worden toegepast op meerdere tekensoorten en verschillende levensfasen.Afgezien van tekendissecties of de toepassing op gastheer-parasietint...

Video: Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetics

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Immunology and Infection

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

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