Impact of pre-analytical handling on bone marrow mRNA gene expression
Stephen Breit
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
These authors contributed equally to this study.
Search for more papers by this authorMatthias Nees
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
These authors contributed equally to this study.
Search for more papers by this authorUlrike Schaefer
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorMargit Pfoersich
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorChristian Hagemeier
Department of Pediatrics, Laboratory for Molecular Biology, Humboldt University, Berlin, Germany
Search for more papers by this authorMartina Muckenthaler
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorAndreas E. Kulozik
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorStephen Breit
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
These authors contributed equally to this study.
Search for more papers by this authorMatthias Nees
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
These authors contributed equally to this study.
Search for more papers by this authorUlrike Schaefer
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorMargit Pfoersich
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorChristian Hagemeier
Department of Pediatrics, Laboratory for Molecular Biology, Humboldt University, Berlin, Germany
Search for more papers by this authorMartina Muckenthaler
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorAndreas E. Kulozik
Department of Paediatric Oncology, Haematology and Immunology, University of Heidelberg, Heidelberg
Search for more papers by this authorSummary
Large clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChipsTM and a custom-designed cDNA array in parallel showed that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18·8%, analysis of variance <0·05), or inconsistent expression that differed from patient to patient (38·4%). Moderate alterations were observed in 42·8% genes, with a maximum fold change <2·0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at −80°C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.
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