Patients with a kidney transplant may encounter chronic dysfunction of their graft. Once damage in the graft has established, therapeutic intervention is less efficient. Clinical parameters and morphologic evaluation of biopsies are used for determining diagnosis and prognosis of the patient. Quantitative polymerase chain reaction (qPCR) may be integrated in clinical practice to facilitate routine diagnostics, risk assessment with respect to graft outcome, and determination of the response to therapy by the patient. The success of qPCR assays is highly dependent on the adequacy of the methodological procedures performed. Here, we describe tips and tricks for processing patient material, RNA analysis, and qPCR primer design and gene expression analyses.
mRNA Transplant Kidney Diagnosis Prognosis
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Part of the results shown in Fig. 5 were derived from a project on B cells in kidney transplantation, initiated by Dr. Sebastiaan Heidt (Leiden University Medical Center, Department of Immunohematology).
Donauer J, Rumberger B, Klein M et al (2003) Expression profiling on chronically rejected transplant kidneys. Transplantation 76:539–547PubMedCrossRefGoogle Scholar
Koop K, Bakker RC, Eikmans M et al (2004) Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA. Kidney Int 66:2038–2046PubMedCrossRefGoogle Scholar
Reeve J, Einecke G, Mengel M et al (2009) Diagnosing rejection in renal transplants: a comparison of molecular- and histopathology-based approaches. Am J Transplant 9:1802–1810PubMedCrossRefGoogle Scholar
Eikmans M, Sijpkens YW, Baelde HJ et al (2002) High transforming growth factor-beta and extracellular matrix mRNA response in renal allografts during early acute rejection is associated with absence of chronic rejection. Transplantation 73:573–579PubMedCrossRefGoogle Scholar
Sarwal M, Chua MS, Kambham N et al (2003) Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. N Engl J Med 349:125–138PubMedCrossRefGoogle Scholar
Mengel M, Reeve J, Bunnag S et al (2009) Molecular correlates of scarring in kidney transplants: the emergence of mast cell transcripts. Am J Transplant 9:169–178PubMedCrossRefGoogle Scholar
Sis B, Jhangri GS, Bunnag S et al (2009) Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am J Transplant 9:2312–2323PubMedCrossRefGoogle Scholar
Rekers NV, Bajema IM, Mallat MJ et al (2012) Quantitative polymerase chain reaction profiling of immunomarkers in rejecting kidney allografts for predicting response to steroid treatment. Transplantation 94:596–602PubMedCrossRefGoogle Scholar
Rekers NV, Bajema IM, Mallat MJ et al (2013) Increased metallothionein expression reflects steroid resistance in renal allograft recipients. Am J Transplant 13(8):2106–2118PubMedCrossRefGoogle Scholar
Desvaux D, Schwarzinger M, Pastural M et al (2004) Molecular diagnosis of renal-allograft rejection: correlation with histopathologic evaluation and antirejection-therapy resistance. Transplantation 78:647–653PubMedCrossRefGoogle Scholar
Flechner SM, Kurian SM, Solez K et al (2004) De novo kidney transplantation without use of calcineurin inhibitors preserves renal structure and function at two years. Am J Transplant 4:1776–1785PubMedCrossRefGoogle Scholar
Roos-Van Groningen MC, Scholten EM, Lelieveld PM et al (2006) Molecular comparison of calcineurin inhibitor-induced fibrogenic responses in protocol renal transplant biopsies. J Am Soc Nephrol 17:881–888PubMedCrossRefGoogle Scholar
Brouard S, Mansfield E, Braud C et al (2007) Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance. Proc Natl Acad Sci U S A 104:15448–15453PubMedCentralPubMedCrossRefGoogle Scholar
Martinez-Llordella M, Lozano JJ, Puig-Pey I et al (2008) Using transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant recipients. J Clin Invest 118:2845–2857PubMedCentralPubMedGoogle Scholar
Sagoo P, Perucha E, Sawitzki B et al (2010) Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans. J Clin Invest 120:1848–1861PubMedCentralPubMedCrossRefGoogle Scholar
Bohne F, Martinez-Llordella M, Lozano JJ et al (2012) Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest 122:368–382PubMedCentralPubMedCrossRefGoogle Scholar
Eikmans M, Roelen DL, Claas FH (2008) Molecular monitoring for rejection and graft outcome in kidney transplantation. Expert Opin Med Diagn 2:1365–1379PubMedCrossRefGoogle Scholar
Eikmans M, Rekers NV, Anholts JD et al (2013) Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation. Blood 121:e81–e89PubMedCrossRefGoogle Scholar