{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T22:31:53Z","timestamp":1781562713029,"version":"3.54.5"},"reference-count":36,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2025,4,26]],"date-time":"2025-04-26T00:00:00Z","timestamp":1745625600000},"content-version":"vor","delay-in-days":25,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2025,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Melanoma invasion of regional lymph nodes, a critical event in the progression of this disease, is well documented as a poor prognostic factor for patients with melanoma. Assessing lymph node involvement is therefore a routine part of the diagnostic workup for patients presenting with melanoma at a T stage of \u2265T2a. In clinical settings, the status and degree of melanoma lymph node involvement is traditionally characterized by histopathological analysis of tissue obtained during a sentinel lymph node biopsy, a labor\u2010intensive and costly approach that requires technically challenging sample preparation and interpretation by a trained pathologist. Alternative approaches that might reduce the financial burden and turnaround time of a sentinel lymph node biopsy workup are therefore desirable. Likewise, the ability to accurately assess lymph node invasion by melanoma in experimental settings is necessary to gain new insights into mechanisms of distant metastasis and tumor\u2010associated immune suppression. With these applications in mind, we recently developed, and describe herein, a tissue\u2010specific qRT\u2010PCR\u2010based protocol for detecting melanoma cells within tumor\u2010draining lymph nodes. Using murine models of lymph\u2010node\u2010invasive and lymph\u2010node\u2010noninvasive melanoma cell lines and a melanin\u2010biosynthesis\u2010pathway\u2010specific <jats:italic>Trp2<\/jats:italic> gene expression assay, we validated this method as a highly sensitive strategy for assessing lymph node involvement by melanoma. \u00a9 2025 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Growth and maintenance of murine melanoma cell cultures<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Extraction and quantification of RNA from murine melanoma cultures<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: cDNA synthesis via reverse transcription<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Probe and primer design for TaqMan\u2010based qPCR<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: qPCR analysis of tissue\u2010specific <jats:italic>Trp2<\/jats:italic> gene expression<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: <jats:italic>In vitro<\/jats:italic> validation of qRT\u2010PCR <jats:italic>Trp2<\/jats:italic> gene expression assay for detection of melanoma cells in murine whole lymph node preparations<\/jats:p><jats:p><jats:bold>Basic Protocol 7<\/jats:bold>: <jats:italic>In vivo<\/jats:italic> validation of qRT\u2010PCR <jats:italic>Trp2<\/jats:italic> gene expression assay for detection of melanoma cells in murine tumor\u2010draining lymph nodes<\/jats:p>","DOI":"10.1002\/cpz1.70139","type":"journal-article","created":{"date-parts":[[2025,4,26]],"date-time":"2025-04-26T15:43:54Z","timestamp":1745682234000},"source":"Crossref","is-referenced-by-count":1,"title":["A Highly Sensitive Tissue\u2010specific qRT\u2010PCR\u2010based Assay for Detection of Melanoma Cells in Tumor\u2010Draining Lymph Nodes"],"prefix":"10.1002","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8668-5228","authenticated-orcid":false,"given":"Kristian M.","family":"Hargadon","sequence":"first","affiliation":[{"name":"Hargadon Laboratory, Department of Biology Hampden\u2010Sydney College  Hampden\u2010Sydney Virginia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Travis B.","family":"Goodloe","sequence":"additional","affiliation":[{"name":"Hargadon Laboratory, Department of Biology Hampden\u2010Sydney College  Hampden\u2010Sydney Virginia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,4,26]]},"reference":[{"key":"e_1_2_13_1_2_1","doi-asserted-by":"publisher","DOI":"10.1001\/JAMADERMATOL.2022.0160"},{"key":"e_1_2_13_1_3_1","doi-asserted-by":"publisher","DOI":"10.1097\/DAD.0000000000000362"},{"key":"e_1_2_13_1_4_1","doi-asserted-by":"publisher","DOI":"10.18632\/oncotarget.10140"},{"key":"e_1_2_13_1_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/jso.24088"},{"key":"e_1_2_13_1_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms4530"},{"key":"e_1_2_13_1_7_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMOA1613210\/SUPPL_FILE\/NEJMOA1613210_DISCLOSURES.PDF"},{"key":"e_1_2_13_1_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellimm.2011.10.003"},{"key":"e_1_2_13_1_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/CNR2.70145"},{"key":"e_1_2_13_1_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.intimp.2018.06.001"},{"key":"e_1_2_13_1_11_1","doi-asserted-by":"publisher","DOI":"10.1080\/2162402X.2015.1069462"},{"key":"e_1_2_13_1_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/modpathol.3801008"},{"key":"e_1_2_13_1_13_1","doi-asserted-by":"publisher","DOI":"10.1001\/JAMASURG.2013.1335"},{"issue":"1","key":"e_1_2_13_1_14_1","first-page":"301","article-title":"Prognostic significance of microscopic tumor burden in sentinel lymph node in patients with cutaneous melanoma","volume":"35","author":"Kim C.","year":"2015","journal-title":"Anticancer Research"},{"issue":"2","key":"e_1_2_13_1_15_1","first-page":"247","article-title":"Immunologic evaluation of spontaneous regression of an intraocular murine melanoma","volume":"31","author":"Knisely T. 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