Preanalytical mRNA stabilization of whole bone marrow samples.
BACKGROUND
Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation.


METHODS
We investigated the effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgene Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens.


RESULTS
Although the overall RNA yield (normalized to 1 x 10(7) leukocytes) was not different, the RNA preparation using unstabilized reference samples had an approximately 3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression (DeltaDeltaC(T) 0.16-0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure (DeltaDeltaC(T) 1.07 and 1.32).


CONCLUSIONS
The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.
Langebrake
Claudia
C
Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany. claudia@langebrake.de
Günther
Kalle
K
Lauber
Jürgen
J
Reinhardt
Dirk
D
D000208:Acute Disease; D001853:Bone Marrow / Q000737:chemistry*; D002648:Child; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics; D050982:GATA1 Transcription Factor / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D019006:Neural Cell Adhesion Molecules / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics; D000099232:Proto-Oncogene Protein Spi-1
D050676:Core Binding Factor Alpha 2 Subunit; D050982:GATA1 Transcription Factor; D019006:Neural Cell Adhesion Molecules; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C494688:GATA1 protein, human; C493728:RUNX1 protein, human