title: Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.
abstract: Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA.
authors.affiliation: INSERM U1155, Hôpital Tenon, Paris, France.|Université Pierre et Marie Curie, Paris 6, Sorbonne Universités, Paris, France.|Urgences Néphrologiques et Transplantation Rénale, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: P
authors.identifier: 0000-0002-2771-1596
authors.initials: P
authors.lastname: Galichon
authors.affiliation: INSERM U1151, Hôpital Necker, Paris, France.
authors.forename: L
authors.initials: L
authors.lastname: Amrouche
authors.affiliation: INSERM U1155, Hôpital Tenon, Paris, France.|Université Pierre et Marie Curie, Paris 6, Sorbonne Universités, Paris, France.|Urgences Néphrologiques et Transplantation Rénale, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Hertig
authors.affiliation: INSERM U1155, Hôpital Tenon, Paris, France.|Université Pierre et Marie Curie, Paris 6, Sorbonne Universités, Paris, France.|Service d'anatomie pathologique, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: I
authors.initials: I
authors.lastname: Brocheriou
authors.affiliation: Laboratoire d'anatomie pathologique, Hôpital Necker, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Rabant
authors.affiliation: INSERM U1155, Hôpital Tenon, Paris, France.
authors.forename: Y-C
authors.initials: YC
authors.lastname: Xu-Dubois
authors.affiliation: Urgences Néphrologiques et Transplantation Rénale, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: N
authors.initials: N
authors.lastname: Ouali
authors.affiliation: Service de Néphrologie et Dialyses, Hôpital Necker, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: K
authors.initials: K
authors.lastname: Dahan
authors.affiliation: Service de Néphrologie et Transplantation Adulte, Hôpital Necker, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: L
authors.initials: L
authors.lastname: Morin
authors.affiliation: INSERM U1151, Hôpital Necker, Paris, France.
authors.forename: F
authors.initials: F
authors.lastname: Terzi
authors.affiliation: INSERM U1155, Hôpital Tenon, Paris, France.|Université Pierre et Marie Curie, Paris 6, Sorbonne Universités, Paris, France.|Urgences Néphrologiques et Transplantation Rénale, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, Paris, France.
authors.forename: E
authors.initials: E
authors.lastname: Rondeau
authors.affiliation: INSERM U1151, Hôpital Necker, Paris, France.|Service de Néphrologie et Transplantation Adulte, Hôpital Necker, Assistance Publique-Hôpitaux de Paris, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.|RTRS "Centaure", Labex "Transplantex", Hôpital Necker, Paris, France.
authors.forename: D
authors.initials: D
authors.lastname: Anglicheau
mesh_terms: D015415:Biomarkers / Q000652:urine*; D005260:Female; D005500:Follow-Up Studies; D005919:Glomerular Filtration Rate; D006084:Graft Rejection / Q000175:diagnosis* / Q000209:etiology / Q000652:urine; D006801:Humans; D007676:Kidney Failure, Chronic / Q000601:surgery*; D007677:Kidney Function Tests; D016030:Kidney Transplantation / Q000009:adverse effects*; D008297:Male; D016133:Polymerase Chain Reaction / Q000592:standards*; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics / Q000652:urine*; D012015:Reference Standards; D012189:Retrospective Studies; D012307:Risk Factors; D014184:Transplantation, Homologous
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger
keywords: clinical research/practice; diagnostic techniques and imaging; kidney transplantation/nephrology; molecular biology; molecular biology: mRNA/mRNA expression; rejection: acute; translational research/science