Performance
The PAXgene Bone Marrow RNA Kit enables efficient isolation and purification of intracellular RNA from heterogeneous human whole bone marrow samples that have been stabilized in the PAXgene Bone Marrow RNA Tube (Figure 1). The process includes an optimized procedure for cell-rich, viscous samples. RNA can be purified days after sample stabilization in PAXgene Bone Marrow RNA Tubes An integrated DNase treatment step ensures that genomic DNA is depleted from samples. In-house and external studies have shown that genomic DNA was <0.85% in 95% of the samples tested (n = 157). A260/A280 values are typically between 1.8 and 2.2, indicating the high purity of the RNA. Combining the PAXgene Bone Marrow RNA Kit with PAXgene Bone Marrow RNA Tubes ensures that RNA is protected from degradation by RNases. Furthermore, stabilization enables accurate gene expression analysis because ex vivo changes in transcript levels due to gene regulation and RNA degradation are minimized (Figure 2).
Principle
Copy numbers of individual mRNA species in bone marrow can change significantly during storage or transport at ambient temperature, making reliable studies of gene expression impossible. Both stabilization of RNA molecules after collection and an efficient RNA purification protocol are needed to maximize insights into expression profiles of bone marrow aspirates. Based on silica-membrane technology in a spin-column format, the PAXgene Bone Marrow RNA Kit provides the chemistry and rapid procedure to isolate RNA of high quality and purity from bone marrow aspirate samples stabilized in PAXgene Bone Marrow RNA Tubes.
Procedure
A bone marrow aspirate sample (2 mL) is introduced into the PAXgene Bone Marrow RNA Tube and incubated at room temperature for a minimum of 2 hours or up to 3 days. Nucleic acids are concentrated by centrifugation and the resuspended precipitate is treated with proteinase K. RNA is then allowed to bind to the silica membrane and contaminants are removed by washing. A DNase treatment step subsequently removes DNA, leaving pure RNA for final elution (Figure 3).
Applications
RNA purified using the PAXgene Bone Marrow RNA Kit is ready for use in a wide range of downstream applications, including:
- cDNA synthesis
- Gene expression arrays
- End-point RT-PCR
- Quantitative RT-PCR, including TaqMan technology
![Bone marrow samples were collected from 39 individuals and immediately transferred to [A] PAXgene Bone Marrow RNA Tubes or [B] EDTA tubes. RNA was purified using the PAXgene Bone Marrow RNA Kit or a reference silica-membrane-based method at day 0 or after 2 days storage at room temperature. Expression levels of five (5) marker transcripts for each sample (IL-8, p53, cox-2, IL-1β, c-fos) were quantified using real-time RT-PCR. The high correlation of CT values (threshold cycles) for the samples stabilized in PAXgene Bone Marrow RNA Tubes indicates that the levels of these transcripts did not significantly change during storage, in contrast to the samples stored in EDTA tubes.](/storage/images/_processed_/e/b/csm_RNA-stability-at-room-temperature_4baca247d2.jpg "Figure 6. RNA stability at room temperature.")
![Bone Marrow samples were stored in PAXgene Bone Marrow RNA Tubes [A] for 3 days at 18-25°C, [B] for 5 days at 2-8°C, or [C] subjected to 2 freeze-thaw cycles. RNA was purified using the PAXgene Bone Marrow RNA Kit, and RNA purity was analyzed by absorbance. The A260/A280 ratios indicate highly pure RNA from samples stored under different conditions.](/storage/images/_processed_/2/9/csm_RNA-Purity-for-samples-stored-under-different-conditions_8f848c41ea.jpg "Figure 7. RNA purity for samples stored under different conditions.")
