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
|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)|
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 and 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.
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.
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.
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.
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.
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.
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.
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 (see RNA yields from bone marrow samples). 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 (see Stability of 5 transcripts at room temperature).
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 and in 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.
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 two hours or up to three 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 (see PAXgene Bone Marrow RNA procedure).
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
|PAXgene Bone Marrow RNA Kit Handbook - For isolation and purification of intracellular RNA from whole bone marrow samples stabilized in PAXgene Bone Marrow RNA Tubes||478 KB|
PAXgene Bone Marrow RNA Kit
1. Can the PAXgene Bone Marrow RNA Kit be used for diagnostic or prognostic procedures?
No. The product is ‘For Research Use.’ Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
2. What RNA yields are expected with this product?
Bone marrow samples are extremely heterogeneous, consisting of varying amounts of cells, tissue, and bone fragments. RNA yields are highly donor-dependent and can vary greatly from sample to sample, as well as between replicates from the same donor, due to sample-intrinsic heterogeneity.
In a study of 295 bone marrow samples, yields of RNA isolated from 2 ml human bone marrow were between 2.0 and 44.7 µg (5-95% quantiles, mean = 15.8 µg, median = 11.0 µg).
3. Can the PAXgene Bone Marrow RNA System be used to isolate viral RNA?
No. The PAXgene Bone Marrow RNA System has been optimized only for cellular RNA.
4. Can DNA be isolated with the kit as well?
No. The PAXgene Bone Marrow RNA System has been optimized only for cellular RNA.
5. Can specific types of white blood cells be enriched from a PAXgene Bone Marrow RNA Tube prior to the RNA isolation?
No. The cells are lysed in the PAXgene Bone Marrow RNA Tube. There is no method available to enrich certain sub-populations of cells after bone marrow is transferred to the PAXgene Bone Marrow RNA Tube.
6. Is RNA from red blood cells also isolated when RNA is extracted from a PAXgene Bone Marrow RNA Tube?
Yes. The PAXgene Bone Marrow RNA System purifies RNA from whole bone marrow. Therefore it also purifies RNA from other cells, e.g. reticulocytes and RBCs. Array analyses have shown higher globin mRNA levels for RNA isolated from whole bone marrow compared to RNA isolated from mononuclear cells.
7. Can bone marrow from leukemia patients with elevated numbers of white blood cells be processed?
Yes. We have tested the system with bone marrow samples containing very high numbers of leukocytes. Bone marrow samples with higher leukocyte numbers are indicated by a highly viscous solution after addition of buffers BMR1 and BMR2 followed by incubation. To obtain high-quality RNA, these samples are further diluted according to directions in the protocol. Diluting these samples accordingly eliminates problems in the extraction procedure.
8. Can I use water to elute the RNA?
Yes. Heating temperature and incubation time will vary depending on different RT protocols. Therefore, the final heating step should be performed routinely at the end of the RNA purification protocol.
9. As part of my RT procedure I routinely heat an aliquot of the eluate prior to the RT reaction. Do I still need to heat the whole eluate after the RNA preparation?
No. An A260/A230 nm absorbance ratio is not specified for any PAXgene Bone Marrow RNA extraction kit. In contrast to a reduced A260/A280 ratio, which results from protein contamination of the eluted RNA and can lead to inhibitory effects in downstream applications, there is no negative impact of a reduced A260/A230 ratio on any downstream application reported to date and therefore, it is not necessary to specify this.
In addition, because the elution buffer contains very low concentrations of salt that absorb at wavelengths between 220 and 230 nm, it is necessary to zero the photometer properly. Do this by preparing a solution for zeroing the photometer which contains a similar salt concentration as the sample to be measured by using the same volumes. Add the same volume of Buffer BMR5 (as the volume of RNA sample to be diluted) into a fresh volume of the buffer in which you will measure the absorption (we recommend 10 mM Tris Cl pH 7.5 buffer for this purpose).
10. Is a A260/A230 nm absorbance ratio specified for RNA derived from the PAXgene Bone Marrow RNA System?
Yes. You may dispose of the waste product from the PAXgene Bone Marrow RNA Tube with a 5% sodium hypochlorite bleach solution, in a 1:9 ratio (1 part bleach : 9 parts tube contents).
11. Can the PAXgene RNA stabilization solution/bone marrow mixture from the PAXgene Bone Marrow RNA Tube be disposed of by adding bleach?
We recommend classifying the tube-processed supernatant as both a biological and chemical waste. Supernatants can be decontaminated by autoclaving and disposed of as chemical waste.
CAUTION: Do not add bleach or acidic solutions directly to the sample preparation waste. Disposal should be done in accordance with local, state, and federal regulations.
12. How are the supernatants resulting from the first two spin steps of the purification procedure disposed since they still may contain biologically hazardous material?
Waste from the sample preparation, such as supernatants from centrifugation steps in the RNA purification procedure, is to be considered potentially infectious. Before disposal, the waste must be autoclaved or incinerated to destroy any infectious material. Disposal must follow official regulations.
13. What is proper disposal procedure for waste from the sample preparation?
|Product||Catalog Nr.||Price (USD)|
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.