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PAXgene Bone Marrow RNA Kit

For purification of intracellular RNA from bone marrow samples in PAXgene Bone Marrow RNA Tubes

  • To be used in conjunction with the PAXgene Bone Marrow RNA Tube
  • Rapid purification of RNA with high integrity and purity
  • Optimized protocol for heterogeneous bone marrow samples
  • Integrated DNase treatment for gDNA removal

Feature

Specification

Format

Spin column

Technology

Silica membrane

Sample amount

2 ml

Elution volume

80 µl (2 x 40 µl)

Main sample type

Bone marrow aspirates

Time per run

95 min/12 samples

Median RNA yield

11 µg/2 ml sample (n=295)

RNA purity

1.8–2.2 (A260/A280)

Processing

Manual: centrifugation


Intended Use

The PAXgene Bone Marrow RNA System consists of a bone marrow collection tube (PAXgene Bone Marrow RNA Tube) and nucleic acid preparation kit (PAXgene Bone Marrow RNA Kit). It is intended for the collection, storage and transport of human bone marrow, stabilization of intracellular RNA in a closed tube and subsequent isolation and purification of total RNA.

For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.

Order information

Product

Catalog Nr.

Price

PAXgene Bone Marrow RNA Kit (30)

For 30 RNA preps: 30 PAXgene Spin Columns, 30 PAXgene Shredder Spin Columns, Processing Tubes, RNase-free DNase I, RNase-free reagents and buffers. To be used in conjunction with PAXgene Bone Marrow RNA Tubes.

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Details

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 stabilized in PAXgene Bone Marrow RNA Tubes, and RNA was purified using the PAXgene Bone Marrow RNA Kit. Yields are indicated for 295 samples, from both in-house and external studies. The median yield for the 295 samples was 11.0 μg RNA per 2 ml sample. Note that RNA yields can vary greatly due to the extreme heterogeneity of bone marrow samples.Zoom image

Figure 1. RNA yields from bone marrow samples.

Bone marrow samples were stabilized in PAXgene Bone Marrow RNA Tubes, and RNA was ...
Bone marrow samples were collected from 39 individuals and immediately transferred and stored in PAXgene Bone Marrow RNA Tubes or EDTA tubes. RNA was purified from the samples on day 0 and day 2 using the PAXgene Bone Marrow RNA Kit. Changes in mRNA levels (ΔCT) from day 0 to day 2 were quantified for each of 5 transcripts. The minimal changes and small standard deviation for samples stabilized in PAXgene Bone Marrow RNA Tubes contrast with the significant changes for samples stored in EDTA tubes.Zoom image

Figure 2. Stability of 5 transcripts at room temperature.

Bone marrow samples were collected from 39 individuals and immediately transferred ...
Zoom image

Figure 3. PAXgene Bone Marrow RNA procedure.

Bone marrow samples were collected in duplicate and stabilized in PAXgene Bone Marrow RNA Tubes. RNA was purified from the samples using the PAXgene Bone Marrow RNA Kit with no storage or after 5 days storage at 2–8°C. RNA integrity was evaluated using the RNA integrity numbers (RIN), measured on the Agilent 2100 Bioanalyzer. Results are shown for three (3) representative donors. The RIN score did not change significantly during storage, indicating that the RNA remains stable in the sample.Zoom image

Figure 4. RNA stabilized at 2–8°C.

Bone marrow samples were collected in duplicate and stabilized in PAXgene Bone ...
Bone marrow samples were stabilized in PAXgene Bone Marrow RNA Tubes, and RNA was purified using the PAXgene Bone Marrow RNA Kit after the indicated number of freeze–thaw cycles. The RIN scores did not change significantly, indicating that RNA remained intact in the samples up to two (2) freeze–thaw cycles.Zoom image

Figure 5. High RNA integrity after freeze–thaw cycles.

Bone marrow samples were stabilized in PAXgene Bone Marrow RNA Tubes, and RNA was ...
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.Zoom image

Figure 6. RNA stability at room temperature.

Bone marrow samples were collected from 39 individuals and immediately transferred ...
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.Zoom image

Figure 7. RNA purity for samples stored under different conditions.

Bone Marrow samples were stored in PAXgene Bone Marrow RNA Tubes [A] for 3 days at ...

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