{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T14:03:59Z","timestamp":1792505039149,"version":"4.5.0"},"reference-count":26,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2017,12,8]],"date-time":"2017-12-08T00:00:00Z","timestamp":1512691200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2017,12,8]],"date-time":"2017-12-08T00:00:00Z","timestamp":1512691200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Subtle molecular differences indicate the heterogeneity present in a number of disease settings. Digital-PCR (dPCR) platforms achieve the necessary levels of sensitivity and accuracy over standard quantitative RT-PCR (qPCR) that promote their use for such situations, detecting low abundance transcript and subtle changes from gene expression. An underlying requisite is good quality RNA, principally dictated by appropriate tissue handling and RNA extraction. Here we consider the application of dPCR to measures of gene expression in pathological tissues with inherent necrosis, focusing on rheumatoid subcutaneous nodules. Variable RNA fragmentation is a feature of RNA from such tissues. Increased presence of transcript fragmentation is reflected in a proportionate decrease in Agilent DV\n                    <jats:sub>200<\/jats:sub>\n                    metric and downstream, a reduction in endogenous control genes\u2019 expression, measured by RT-dPCR. We show that normalisation of target gene expression to that for endogenous control genes sufficiently corrects for the variable level of fragmented RNA. Recovery of target gene values was achieved in samples comprising as much as 50 percent fragmented RNA, indicating the suitability and appropriate limitation of such data treatment when applied to samples obtained from inherently necrotic tissues.\n                  <\/jats:p>","DOI":"10.1038\/s41598-017-17619-0","type":"journal-article","created":{"date-parts":[[2017,12,4]],"date-time":"2017-12-04T06:59:04Z","timestamp":1512370744000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Digital-PCR for gene expression: impact from inherent tissue RNA degradation"],"prefix":"10.1038","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4702-6078","authenticated-orcid":false,"given":"Melanie J.","family":"Millier","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lisa K.","family":"Stamp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6972-1242","authenticated-orcid":false,"given":"Paul A.","family":"Hessian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2017,12,8]]},"reference":[{"key":"17619_CR1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-017-02217-x","volume":"7","author":"SC Taylor","year":"2017","unstructured":"Taylor, S. C., Laperriere, G. & Germain, H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep \n                           7, 2409, https:\/\/doi.org\/10.1038\/s41598-017-02217-x (2017).","journal-title":"Sci Rep"},{"key":"17619_CR2","doi-asserted-by":"publisher","DOI":"10.1038\/srep13174","volume":"5","author":"L Dong","year":"2015","unstructured":"Dong, L. et al. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Sci Rep \n                           5, 13174, https:\/\/doi.org\/10.1038\/srep13174 (2015).","journal-title":"Sci Rep"},{"key":"17619_CR3","doi-asserted-by":"publisher","first-page":"e75296","DOI":"10.1371\/journal.pone.0075296","volume":"8","author":"R Sanders","year":"2013","unstructured":"Sanders, R., Mason, D. J., Foy, C. A. & Huggett, J. F. Evaluation of digital PCR for absolute RNA quantification. PLoS One \n                           8, e75296, https:\/\/doi.org\/10.1371\/journal.pone.0075296 (2013).","journal-title":"PLoS One"},{"key":"17619_CR4","doi-asserted-by":"publisher","DOI":"10.1038\/modpathol.2017.82","author":"A Aguilar-Mahecha","year":"2017","unstructured":"Aguilar-Mahecha, A. et al. The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience. Mod Pathol. https:\/\/doi.org\/10.1038\/modpathol.2017.82 (2017).","journal-title":"Mod Pathol"},{"key":"17619_CR5","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1136\/ard.62.1.77","volume":"62","author":"J Highton","year":"2003","unstructured":"Highton, J., Hessian, P. A., Kean, A. & Chin, M. Cell death by apoptosis is a feature of the rheumatoid nodule. Ann Rheum Dis \n                           62, 77\u201380 (2003).","journal-title":"Ann Rheum Dis"},{"key":"17619_CR6","doi-asserted-by":"publisher","first-page":"39676","DOI":"10.18632\/oncotarget.5344","volume":"6","author":"G Bredholt","year":"2015","unstructured":"Bredholt, G. et al. Tumor necrosis is an important hallmark of aggressive endometrial cancer and associates with hypoxia, angiogenesis and inflammation responses. Oncotarget \n                           6, 39676\u201339691, https:\/\/doi.org\/10.18632\/oncotarget.5344 (2015).","journal-title":"Oncotarget"},{"key":"17619_CR7","doi-asserted-by":"publisher","first-page":"1371","DOI":"10.1016\/j.bbabio.2006.06.014","volume":"1757","author":"N Festjens","year":"2006","unstructured":"Festjens, N., Vanden Berghe, T. & Vandenabeele, P. Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim Biophys Acta \n                           1757, 1371\u20131387, https:\/\/doi.org\/10.1016\/j.bbabio.2006.06.014 (2006).","journal-title":"Biochim Biophys Acta"},{"key":"17619_CR8","doi-asserted-by":"publisher","first-page":"1079","DOI":"10.1016\/j.celrep.2015.04.026","volume":"11","author":"MP Thomas","year":"2015","unstructured":"Thomas, M. P. et al. Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3\u2032 Uridylated Intermediates Degraded by DIS3L2. Cell Rep \n                           11, 1079\u20131089, https:\/\/doi.org\/10.1016\/j.celrep.2015.04.026 (2015).","journal-title":"Cell Rep"},{"key":"17619_CR9","doi-asserted-by":"publisher","first-page":"1272","DOI":"10.1101\/gad.1199904","volume":"18","author":"WX Zong","year":"2004","unstructured":"Zong, W. X., Ditsworth, D., Bauer, D. E., Wang, Z. Q. & Thompson, C. B. Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev \n                           18, 1272\u20131282, https:\/\/doi.org\/10.1101\/gad.1199904 (2004).","journal-title":"Genes Dev"},{"key":"17619_CR10","doi-asserted-by":"publisher","DOI":"10.1186\/s12885-016-2197-1","volume":"16","author":"R Narendrula","year":"2016","unstructured":"Narendrula, R. et al. RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines. BMC Cancer \n                           16, 146, https:\/\/doi.org\/10.1186\/s12885-016-2197-1 (2016).","journal-title":"BMC Cancer"},{"key":"17619_CR11","doi-asserted-by":"publisher","first-page":"126","DOI":"10.1016\/j.mam.2005.12.003","volume":"27","author":"S Fleige","year":"2006","unstructured":"Fleige, S. & Pfaffl, M. W. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med \n                           27, 126\u2013139, https:\/\/doi.org\/10.1016\/j.mam.2005.12.003 (2006).","journal-title":"Mol Aspects Med"},{"key":"17619_CR12","doi-asserted-by":"publisher","first-page":"573","DOI":"10.1007\/s00414-011-0578-1","volume":"125","author":"A Koppelkamm","year":"2011","unstructured":"Koppelkamm, A., Vennemann, B., Lutz-Bonengel, S., Fracasso, T. & Vennemann, M. RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays. Int J Legal Med \n                           125, 573\u2013580, https:\/\/doi.org\/10.1007\/s00414-011-0578-1 (2011).","journal-title":"Int J Legal Med"},{"key":"17619_CR13","doi-asserted-by":"publisher","first-page":"e63","DOI":"10.1093\/nar\/gkr065","volume":"39","author":"J Vermeulen","year":"2011","unstructured":"Vermeulen, J. et al. Measurable impact of RNA quality on gene expression results from quantitative PCR. Nucleic Acids Res \n                           39, e63, https:\/\/doi.org\/10.1093\/nar\/gkr065 (2011).","journal-title":"Nucleic Acids Res"},{"key":"17619_CR14","unstructured":"Houlder, E. L. et al. Expression of the genes facilitating methotrexate action within subcutaneous rheumatoid nodules. Clin Exp Rheumatol In Press (2017)."},{"key":"17619_CR15","doi-asserted-by":"publisher","first-page":"1783","DOI":"10.1002\/1529-0131(199810)41:10<1783::AID-ART10>3.0.CO;2-W","volume":"41","author":"R Wikaningrum","year":"1998","unstructured":"Wikaningrum, R. et al. Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient. Arthritis Rheum \n                           41, 1783\u20131797, https:\/\/doi.org\/10.1002\/1529-0131(199810)41:10<1783::AID-ART10>3.0.CO;2-W (1998).","journal-title":"Arthritis Rheum"},{"key":"17619_CR16","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1006\/abio.1998.2914","volume":"265","author":"LJ Jones","year":"1998","unstructured":"Jones, L. J., Yue, S. T., Cheung, C. Y. & Singer, V. L. RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Anal Biochem \n                           265, 368\u2013374, https:\/\/doi.org\/10.1006\/abio.1998.2914 (1998).","journal-title":"Anal Biochem"},{"key":"17619_CR17","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/0090-1229(87)90107-3","volume":"45","author":"DG Palmer","year":"1987","unstructured":"Palmer, D. G., Hogg, N., Allen, C. A., Highton, J. & Hessian, P. A. A mononuclear phagocyte subset associated with cell necrosis in rheumatoid nodules: identification with monoclonal antibody 5.5. Clin Immunol Immunopathol \n                           45, 17\u201328 (1987).","journal-title":"Clin Immunol Immunopathol"},{"key":"17619_CR18","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2199-7-3","volume":"7","author":"A Schroeder","year":"2006","unstructured":"Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol \n                           7, 3, https:\/\/doi.org\/10.1186\/1471-2199-7-3 (2006).","journal-title":"BMC Mol Biol"},{"key":"17619_CR19","doi-asserted-by":"publisher","first-page":"259","DOI":"10.1515\/bmc-2012-0056","volume":"4","author":"MJ Morgan","year":"2013","unstructured":"Morgan, M. J. & Liu, Z. G. Programmed cell death with a necrotic-like phenotype. Biomol Concepts \n                           4, 259\u2013275, https:\/\/doi.org\/10.1515\/bmc-2012-0056 (2013).","journal-title":"Biomol Concepts"},{"key":"17619_CR20","doi-asserted-by":"publisher","first-page":"700","DOI":"10.1038\/nrm2970","volume":"11","author":"P Vandenabeele","year":"2010","unstructured":"Vandenabeele, P., Galluzzi, L., Vanden Berghe, T. & Kroemer, G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol \n                           11, 700\u2013714, https:\/\/doi.org\/10.1038\/nrm2970 (2010).","journal-title":"Nat Rev Mol Cell Biol"},{"key":"17619_CR21","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1038\/cdd.2011.164","volume":"19","author":"N Vanlangenakker","year":"2012","unstructured":"Vanlangenakker, N., Vanden Berghe, T. & Vandenabeele, P. Many stimuli pull the necrotic trigger, an overview. Cell Death Differ \n                           19, 75\u201386, https:\/\/doi.org\/10.1038\/cdd.2011.164 (2012).","journal-title":"Cell Death Differ"},{"key":"17619_CR22","doi-asserted-by":"publisher","first-page":"e0118226","DOI":"10.1371\/journal.pone.0118226","volume":"10","author":"A Zmienko","year":"2015","unstructured":"Zmienko, A. et al. Selection of reference genes for qPCR- and ddPCR-based analyses of gene expression in Senescing Barley leaves. PLoS One \n                           10, e0118226, https:\/\/doi.org\/10.1371\/journal.pone.0118226 (2015).","journal-title":"PLoS One"},{"key":"17619_CR23","doi-asserted-by":"publisher","first-page":"e1261","DOI":"10.1371\/journal.pone.0001261","volume":"2","author":"S von Ahlfen","year":"2007","unstructured":"von Ahlfen, S., Missel, A., Bendrat, K. & Schlumpberger, M. Determinants of RNA quality from FFPE samples. PLoS One \n                           2, e1261, https:\/\/doi.org\/10.1371\/journal.pone.0001261 (2007).","journal-title":"PLoS One"},{"key":"17619_CR24","doi-asserted-by":"publisher","first-page":"e0165181","DOI":"10.1371\/journal.pone.0165181","volume":"11","author":"RF Walter","year":"2016","unstructured":"Walter, R. F. et al. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology. PLoS One \n                           11, e0165181, https:\/\/doi.org\/10.1371\/journal.pone.0165181 (2016).","journal-title":"PLoS One"},{"key":"17619_CR25","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1002\/art.1780310302","volume":"31","author":"FC Arnett","year":"1988","unstructured":"Arnett, F. C. et al. The American Rheumatism Association1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum \n                           31, 315\u2013324 (1988).","journal-title":"Arthritis Rheum"},{"key":"17619_CR26","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2007-8-2-r19","volume":"8","author":"J Hellemans","year":"2007","unstructured":"Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol \n                           8, R19, https:\/\/doi.org\/10.1186\/gb-2007-8-2-r19 (2007).","journal-title":"Genome Biol"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-17619-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-17619-0","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-17619-0.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,23]],"date-time":"2022-12-23T10:48:09Z","timestamp":1671792489000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-017-17619-0"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,12,8]]},"references-count":26,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2017,12]]}},"alternative-id":["17619"],"URL":"https:\/\/doi.org\/10.1038\/s41598-017-17619-0","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,12,8]]},"assertion":[{"value":"11 October 2017","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 November 2017","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"8 December 2017","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare that they have no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"17235"}}