RNA isolation from Peyer
Main Article Content
Keywords
B-lymphocytes, mononuclear phagocytes, Peyer, Nanostring technology, T-lymphocytes.
Abstract
Sampling and immune surveillance within gut-associated lymphoid tissues (GALT) such as the intestinal Peyer’s patch (PP) occurs by an elegantly orchestrated effort that involves the epithelial barrier, B and T lymphocytes, and an extensive network of mononuclear phagocytes. Although we now understand more about the dynamics of antigen and microbial sampling within PPs, the gene expression changes that occur in individual cell subsets during sampling are not well characterized. This protocol describes the isolation of high-quality RNA from sorted PP, B and T-lymphocytes, and CD11c+ phagocytes for use with nCounter-nanostring technology. This method allows investigators to study gene expression changes within PPs in response to antigens, microbes, and oral vaccine delivery vehicles of interest that are sampled.
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References
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2. Didierlaurent A, Sirard JC, Kraehenbuhl JP, Neutra MR (2002) How the gut senses its content. Cell Microbiol 4: 61-72.
3. Kanaya T, Ohno H (2014) The Mechanisms of M-cell Differentiation. Biosci Microbiota Food Health 33: 91-97.
4. Kraehenbuhl JP, Neutra MR (2000) Epithelial M cells: differentiation and function. Annu Rev Cell Dev Biol 16: 301-332.
5. Ohno H (2016) Intestinal M cells. J Biochem 159: 151-160.
6. Mabbott NA, Donaldson DS, Ohno H, Williams IR, Mahajan A (2013) Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol 6: 666-677.
7. Iwasaki A, Kelsall BL (2001) Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells. J Immunol 166: 4884-4890.
8. Rochereau N, Verrier B, Pin JJ, Genin C, Paul S (2011) Phenotypic localization of distinct DC subsets in mouse Peyer Patch. Vaccine 29: 3655-3661.
9. Lelouard H, Henri S, De Bovis B, Mugnier B, Chollat-Namy A, et al. (2010) Pathogenic bacteria and dead cells are internalized by a unique subset of Peyer's patch dendritic cells that express lysozyme. Gastroenterology 138: 173-184 e171-173.
10. De Jesus M, Ostroff GR, Levitz SM, Bartling TR, Mantis NJ (2014) A population of Langerin-positive dendritic cells in murine Peyer's patches involved in sampling beta-glucan microparticles. PLoS One 9: e91002.
11. Reboldi A, Arnon TI, Rodda LB, Atakilit A, Sheppard D, et al. (2016) IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches. Science 352: aaf4822.
12. Salazar-Gonzalez RM, Niess JH, Zammit DJ, Ravindran R, Srinivasan A, et al. (2006) CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer's patches. Immunity 24: 623-632.
13. De Jesus M, Rodriguez AE, Yagita H, Ostroff GR, Mantis NJ (2015) Sampling of Candida albicans and Candida tropicalis by Langerin-positive dendritic cells in mouse Peyer's patches. Immunol Lett 168: 64-72.
14. Bonnardel J, Da Silva C, Wagner C, Bonifay R, Chasson L, et al. (2017) Distribution, location, and transcriptional profile of Peyer's patch conventional DC subsets at steady state and under TLR7 ligand stimulation. Mucosal Immunol 10: 1412-1430.
15. Da Silva C, Wagner C, Bonnardel J, Gorvel JP, Lelouard H (2017) The Peyer's Patch Mononuclear Phagocyte System at Steady State and during Infection. Front Immunol 8: 1254.
16. Bonnardel J, Da Silva C, Masse M, Montanana-Sanchis F, Gorvel JP, et al. (2015) Gene expression profiling of the Peyer's patch mononuclear phagocyte system. Genom Data 5: 21-24.
17. Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, et al. (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26: 317-325.
18. Huang H, Ostroff GR, Lee CK, Specht CA, Levitz SM (2013) Characterization and optimization of the glucan particle-based vaccine platform. Clin Vaccine Immunol 20: 1585-1591.
19. Young SH, Ostroff GR, Zeidler-Erdely PC, Roberts JR, Antonini JM, et al. (2007) A comparison of the pulmonary inflammatory potential of different components of yeast cell wall. J Toxicol Environ Health A 70: 1116-1124.
20. Mirza Z, Soto ER, Dikengil F, Levitz SM, Ostroff GR (2017) Beta-Glucan Particles as Vaccine Adjuvant Carriers. Methods Mol Biol 1625: 143-157.
21. De Jesus M, Ahlawat S, Mantis NJ (2013) Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches. J Vis Exp.
22. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550.
23. Chen J, Tambalo M, Barembaum M, Ranganathan R, Simoes-Costa M, et al. (2017) A systems-level approach reveals new gene regulatory modules in the developing ear. Development 144: 1531-1543.
24. De Jesus M, Ahlawat S, Mantis NJ (2013) Isolating and immunostaining lymphocytes and dendritic cells from murine Peyer's patches. J Vis Exp: e50167.
25. Dudakov JA, Hanash AM, van den Brink MR (2015) Interleukin-22: immunobiology and pathology. Annu Rev Immunol 33: 747-785.
26. Renner M, Bergmann G, Krebs I, End C, Lyer S, et al. (2007) DMBT1 confers mucosal protection in vivo and a deletion variant is associated with Crohn's disease. Gastroenterology 133: 1499-1509.
27. Chen VL, Surana NK, Duan J, Kasper DL (2013) Role of murine intestinal interleukin-1 receptor 1-expressing lymphoid tissue inducer-like cells in Salmonella infection. PLoS One 8: e65405.
28. Kay RA, Ellis IR, Jones SJ, Perrier S, Florence MM, et al. (2005) The expression of migration stimulating factor, a potent oncofetal cytokine, is uniquely controlled by 3'-untranslated region-dependent nuclear sequestration of its precursor messenger RNA. Cancer Res 65: 10742-10749.
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Published
Jul 2, 2018
Article Details
How to Cite
1.
Singh N, Gallagher HC, Song R, Dhinsa JK, Ostroff GR, De Jesus M. RNA isolation from Peyer. J Biol Methods [Internet]. 2018 Jul. 2 [cited 2024 Apr. 16];5(3):e95. Available from: https://jbmethods.org/index.php/jbm/article/view/246
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Author Biography
Magdia De Jesus, Department of Biomedical Sciences, University at Albany, School of Public Health Albany, NY USA and Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany NY USA
Assistant Professor, Biomedical Science
