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In spite of the efforts to clarify the relationship among genetic factors and susceptibility to develop OP, many genetic associations need to be further functionally validated. Besides, some limitations as the choice of stably expressed reference genes (RG) should be overcome to ensure the quality and reproducibility of gene expression assays. To our knowledge, a validation study for RG in OP is still missing. We compared the expression levels, using polymerase chain reaction quantitative real time (qPCR) of 10 RG (<jats:italic>G6PD<\/jats:italic>, <jats:italic>B2M<\/jats:italic>, <jats:italic>GUSB<\/jats:italic>, <jats:italic>HSP90<\/jats:italic>, <jats:italic>EF1A<\/jats:italic>, <jats:italic>RPLP0<\/jats:italic>, <jats:italic>GAPDH<\/jats:italic>, <jats:italic>ACTB<\/jats:italic>, <jats:italic>18\u2009S<\/jats:italic> and <jats:italic>HPRT1<\/jats:italic>) to assess their suitability in OP analysis by using GeNorm, Normfinder, BestKeeper and RefFinder programs. A minimal number of two RG was recommended by GeNorm to obtain a reliable normalization. <jats:italic>RPLP0<\/jats:italic> and <jats:italic>B2M<\/jats:italic> were identified as the most stable genes in OP studies while <jats:italic>ACTB<\/jats:italic>, <jats:italic>18\u2009S<\/jats:italic> and <jats:italic>HPRT1<\/jats:italic> were inadequate for normalization in our data set. Moreover, we showed the dramatic effects of suboptimal RG choice on the quantification of a target gene, highlighting the importance in the identification of the most appropriate reference gene to specific diseases. We suggest the use of <jats:italic>RPLP0<\/jats:italic> and <jats:italic>B2M<\/jats:italic> as the most stable reference genes while we do not recommend the use of the least stable reference genes <jats:italic>HPRT1<\/jats:italic>, <jats:italic>18\u2009S<\/jats:italic> and <jats:italic>ACTB<\/jats:italic> in OP expression assays using PBMC as biological source. Additionally, we emphasize the importance of individualized and careful choice in software and reference genes selection.<\/jats:p>","DOI":"10.1038\/s41598-019-52612-9","type":"journal-article","created":{"date-parts":[[2019,11,11]],"date-time":"2019-11-11T11:03:16Z","timestamp":1573470196000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Postmenopausal Osteoporosis reference genes for qPCR expression assays"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0320-3674","authenticated-orcid":false,"given":"Camilla Albertina Dantas","family":"de Lima","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suelen Cristina","family":"de Lima","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandre Domingues","family":"Barbosa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paula","family":"Sandrin-Garcia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Will","family":"de Barros Pita","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jaqueline","family":"de Azev\u00eado Silva","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8493-1168","authenticated-orcid":false,"given":"Sergio","family":"Crovella","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2019,11,11]]},"reference":[{"key":"52612_CR1","doi-asserted-by":"publisher","first-page":"391","DOI":"10.1590\/2359-3997000000178","volume":"60","author":"MGM Rocha-Braz","year":"2016","unstructured":"Rocha-Braz, M. 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