Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

Abstract

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 ؋ 10 7 leukocytes) was not different, the RNA preparation using unstabilized reference samples had an ϳ3 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 (⌬⌬C 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 (⌬⌬C 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.

Assessment of mRNA and microRNA Stabilization in Peripheral Human Blood for Multicenter Studies and Biobanks

“…Analysis of RNA integrity and purity served as tool to determine the overall quality of stabilization, preparation, and purification that can be influenced by degradation 3. Regarding purity, ratios >1.8 are considered suitable for gene expression analyses 20.…”

“…Study centers face the challenge of having limited access to research laboratory support and might be unable to perform RNA isolation immediately after blood collection 2. Additionally, isolation and analysis of RNA in a central laboratory avoid inter-laboratory variations 3. Such a study design results in unique challenges due to pre-analytical variations occurring before marker analyses and includes factors like method of blood collection, shipping conditions, and time delay between sample collection and analysis 4.…”

Interindividual and Interethnic Variation in Genomewide Gene Expression: Insights into the Biological Variation of Gene Expression and Clinical Implications

“…Similar to the assessments of routinely measured clinical analytes, several aspects of QC have recently been examined for gene expression, including sample processing (6,11,12 ), preanalytical optimization and variation (13)(14)(15), and effects of long-term storage (16 ).…”

Molecular Techniques in Hematopathology

“…The PAXgene series of RNA stabilization tubes [PreAnalytiX; joint venture, Qiagen (Valencia, CA) and Becton-Dickinson (Franklin Lake, NJ)] is widely used and has been shown to reliably maintain RNA integrity [12][13][14][15][16]. Other collection systems are available from Ambion (Austin, TX), Applied Biosystems (Foster City, CA), Promega (Madison, WI), Invitrogen (Carlsbad, CA), Zymo Research (Orange, CA), and Gentra Systems (Plymouth, MN).…”