{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2023,8,26]],"date-time":"2023-08-26T04:26:45Z","timestamp":1693024005615},"reference-count":48,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2006,10,1]],"date-time":"2006-10-01T00:00:00Z","timestamp":1159660800000},"content-version":"vor","delay-in-days":242,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CP Microbiology"],"published-print":{"date-parts":[[2006,2]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>This unit provides a chronological in\u2010depth description of all protocols needed for quantitative reverse transcription\u2013PCR (Q\u2010RT\u2010PCR) analysis of <jats:named-content content-type=\"genus-species\"><jats:italic>Borrelia burgdorferi<\/jats:italic><\/jats:named-content> gene expression within infected mouse tissues. Specifically, this unit discusses the extraction of RNA from infected mouse tissues, removal of contaminating genomic DNA from the purified RNA, preparation of cDNA and genomic DNA standards, LightCycler\u2010based PCR, and a relative quantification analysis of the cDNA. Q\u2010RT\u2010PCR as a highly relevant and powerful tool used to detect gene expression by bacteria within mammalian host tissues. It is also an invaluable technique used to measure and assess the small differences in expression that can exist among a set of genes, whereas this type of analysis is not feasible using less\u2010sensitive techniques such as indirect immunofluorescence analysis or qualitative RT\u2010PCR. Although the protocols described herein are tailored for <jats:named-content content-type=\"genus-species\"><jats:italic>B. burgdorferi<\/jats:italic><\/jats:named-content>, they are broadly applicable to other microbes.<\/jats:p>","DOI":"10.1002\/9780471729259.mc01d03s00","type":"journal-article","created":{"date-parts":[[2005,10,25]],"date-time":"2005-10-25T14:20:34Z","timestamp":1130250034000},"source":"Crossref","is-referenced-by-count":3,"title":["Example of Real\u2010Time Quantitative Reverse Transcription\u2013PCR (Q\u2010RT\u2010PCR) Analysis of Bacterial Gene Expression during Mammalian Infection: <i>Borrelia burgdorferi<\/i> in Mouse Tissues"],"prefix":"10.1002","volume":"00","author":[{"given":"Jennifer C.","family":"Miller","sequence":"first","affiliation":[{"name":"University of Kentucky College of Medicine  Lexington Kentucky"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2006,10]]},"reference":[{"key":"e_1_2_9_2_1_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI2325"},{"key":"e_1_2_9_2_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.1995.mmi_18030507.x"},{"key":"e_1_2_9_2_3_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.69.6.4146-4153.2001"},{"key":"e_1_2_9_2_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.72.11.6433-6445.2004"},{"key":"e_1_2_9_2_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.68.12.6677-6684.2000"},{"key":"e_1_2_9_2_6_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.69.9.5286-5293.2001"},{"key":"e_1_2_9_2_7_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.67.7.3181-3187.1999"},{"key":"e_1_2_9_2_8_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2000.01698.x"},{"key":"e_1_2_9_2_9_1","first-page":"532","article-title":"A reagent for the single\u2010step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples","volume":"15","author":"Chomczynski P.","year":"1993","journal-title":"Biotechniques"},{"key":"e_1_2_9_2_10_1","first-page":"942","article-title":"Modification of the TRI reagent procedure for isolation of RNA from polysaccharide\u2010 and proteoglycan\u2010rich sources","volume":"19","author":"Chomczynski P.","year":"1995","journal-title":"Biotechniques"},{"key":"e_1_2_9_2_11_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1995.1126"},{"key":"e_1_2_9_2_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(87)90021-2"},{"key":"e_1_2_9_2_13_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI119264"},{"key":"e_1_2_9_2_14_1","doi-asserted-by":"publisher","DOI":"10.1038\/37551"},{"key":"e_1_2_9_2_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1286-4579(01)01435-6"},{"key":"e_1_2_9_2_16_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.40.11.4256-4265.2002"},{"key":"e_1_2_9_2_17_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.69.6.3618-3627.2001"},{"key":"e_1_2_9_2_18_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.71.9.5042-5055.2003"},{"key":"e_1_2_9_2_19_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.70.7.3382-3388.2002"},{"key":"e_1_2_9_2_20_1","volume-title":"PCR Protocols: A Guide to Methods and Applications","author":"Kawasaki E.S.","year":"1990"},{"key":"e_1_2_9_2_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00430-004-0218-1"},{"key":"e_1_2_9_2_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0002-9440(10)63359-7"},{"key":"e_1_2_9_2_23_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.69.8.4831-4838.2001"},{"key":"e_1_2_9_2_24_1","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.26120-0"},{"key":"e_1_2_9_2_25_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.182.21.6254-6258.2000"},{"key":"e_1_2_9_2_26_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.38.4.1569-1574.2000"},{"key":"e_1_2_9_2_27_1","unstructured":"Miller J.C. 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