Preanalytical mRNA Stabilization of Whole Bone Marrow Samples.

Background: Gene expression profiling is a useful tool for the diagnosis and basic research of cancer. One of the major limitations of this approach is that already a short-term storage of native specimens of peripheral blood (PB) or bone marrow (BM) and/or the method of RNA isolation has significant influence on gene expression due to gene induction, repression and RNA degradation. The objective of the current study was to investigate the influence of a newly developed RNA stabilization and preparation system for BM specimens (PAXgeneTM Bone Marrow RNA System), focusing on RNA-yield, RNA-integrity and stability of gene expression profiles over time of BM sample storage.

Patients and methods: 180 RNA samples that have been processed from 45 heparinized BM specimens of unselected children with acute leukemia at initial diagnosis, during treatment or without pathological findings in hematopoiesis were analyzed. Immediately after collection, heparinized BM specimens were divided into two PAXgeneTM RNA tubes and two standard tubes, each. RNA isolation using either the PAXgeneTM protocol (P) or a reference protocol (R) was performed after 2 hours (P0 and R0) or after 48 hours (P2 and R2).

Results: The overall RNA yield (normalized to 1×107 leukocytes) was not different in all four isolation procedures, however the integrity of the RNA using the PAXgeneTM system was significantly higher at both time-points than that of the reference system: 8.6±0.2 (P0) vs. 6.8±0.4 (R0), p=0.0003 and 8.1±0.2 (P2) vs. 6.7±0.5 (R2), p=0.008. For the stabilized samples, we found very good pairwise correlation in gene expression for either gene at P2 compared to P0: GATA1 89±6%, RUNX1 83±10%, NCAM 69±12% and SPI1 89±6%. However, there were significant differences in two of the analyzed genes using the reference RNA isolation procedure: 40±6%, p<0.001 (GATA1) and 47±8%, p=0.005 (NCAM), respectively. Comparing the two RNA isolation procedures, there are significant differences in the expression levels of the analyzed genes.

Discussion: The PAXgeneTM system is appropriate for the stabilization of gene expression levels in BM samples after short-term storage. However, as in some genes the reference RNA isolation procedure resulted in similar gene expression levels, the use of a stabilization reagent has to be carefully evaluated within the preanalytical handling of the samples. Our analysis has shown that it is not suitable to compare RNA expression levels derived from different isolation procedures. In conclusion, the PAXgeneTM system is able to stabilize RNA from clinical BM samples while being suitable to isolate high quality and quantity RNA.