PAXgene Blood RNA MDx Kit
For automated, high-throughput purification of cellular RNA from whole blood
- Automated purification of RNA from 48–96 samples
- Standardized sample processing prior to analysis
- Integrated DNase treatment for removal of gDNA
- Isolation of high-quality intracellular RNA
|Sample amount||2.5 ml|
|Elution volume||120 µl|
|Main sample type||Whole blood|
|Time per run||3–4 h|
|Typical RNA yield||>3 µg/2.5 ml sample|
|RNA purity||1.8–2.2 (A260/A280)|
|Processing||Automated: BioRobot Universal System
or BioRobot MDx Workstation
The PAXgene Blood RNA MDx Kit is intended for the purification of RNA from whole blood collected in PAXgene Blood RNA Tubes and processed on the BioRobot Universal System.
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.
Following collection of blood samples (2.5 ml) in PAXgene Blood RNA Tubes, the simple purification procedure begins with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube. Proteinase K and an optimized incubation buffer are added to digest proteins. The tubes are then transferred to the BioRobot MDx or BioRobot Universal System for automated RNA purification.
A total of 48 water samples were processed on the BioRobot MDx workstation using the PAXgene Blood RNA MDx Kit. The eluates were pooled and added to quantitative, real-time RT-PCR assays at the percentage indicated. Each assay was performed in quadruplicate using the same amount of RNA from HeLa cells, with primers and probe for β-actin. [A] CT values are given (mean ± standard deviation), with the difference between the mean CT values and the mean CT value for the 0% reactions (ΔCT). None of the reactions were inhibited (ΔCT ≥0.5). [B] Amplification curves for all 20 assays show no detectable inhibition of RT-PCR.
Blood was collected in duplicate from 3 donors into PAXgene Blood RNA Tubes. RNA was purified using the PAXgene Blood RNA MDx Kit on the BioRobot MDx instrument. RNA was analyzed with an Agilent 2100 Bioanalyzer. The RNA integrity number (RIN), indicated on each scan, shows the high quality of the purified RNA. [A] Analysis of 2 samples from donor A. [B] Analysis of 2 samples from donor B. [C] Analysis of 2 samples from donor C.
RNA was purified from 384 blood samples collected in PAXgene Blood RNA Tubes and processed in 4 runs on the BioRobot MDx using the PAXgene Blood RNA MDx Kit. Genomic DNA was quantified using a genomic-DNA-specific real-time TaqMan PCR assay with primers and probe for the β-actin gene. The amount of DNA present is given as a percentage of the total nucleic acid content.
Blood was collected in fivefold replicates from 6 donors into PAXgene Blood RNA Tubes. RNA was purified using the PAXgene Blood RNA MDx Kit on 3 different BioRobot MDx workstations (MDx 1, MDx 2, MDx 3) and manually using the PAXgene 96 Blood RNA Kit (PAX 96). Purified RNA was analyzed using quantitative, real-time RT-PCR, with primers and probe for the c-fos transcript. All values were well within the ±3x total precision of the assay (2.34 CT), indicated by the horizontal lines.
The automated PAXgene Blood RNA MDx procedure provides high-quality RNA (see High-quality RNA from different donors). An integrated DNase treatment ensures that genomic DNA contamination is minimized, typically to <0.1% (see Very low levels of genomic DNA). The fully automated PAXgene Blood RNA MDx procedure provides RNA yields comparable to the manual PAXgene 96 Blood RNA procedure (typically ≥80%). Typical yields of RNA isolated from 2.5 ml healthy, human, whole blood (4.6 x 106 – 1.1 x 107 leukocytes/ml) are ≥3 µg for >95% of the samples processed. Since yields are highly donor-dependent, individual yields may vary. Purified RNA exhibits no detectable RT-PCR inhibition and intersystem variation is minimal and well within the error range of typical real-time RT-PCR assays (see High intersystem reproducibility).
The robust and standardized procedure is designed to minimize the risk of sample-to-sample cross-contamination. Total RNA was purified from 48 blood samples collected in PAXgene Blood RNA Tubes and 48 water samples. The samples were arranged in a checkerboard pattern on a 96-well block. After purification using the PAXgene Blood RNA MDx Kit on the BioRobot MDx workstation, all samples were analyzed by quantitative, real-time PCR using primers and probes specific for 18s rRNA for 40 PCR cycles. Threshold cycle values (CT) for the blood samples ranged from 27.3 to 32.7 while no RNA was detected in the water samples after 40 cycles.
Copy numbers of individual RNA species in blood can change significantly during storage or transport at ambient temperature, making reliable studies of gene expression difficult. Both stabilization of RNA molecules after collection and an efficient RNA purification protocol are needed to maximize insights into expression profiles of whole blood. Based on proven silica-membrane technology in a high-throughput, 96-well plate format, and when combined with PAXgene Blood RNA Tubes, the PAXgene 96 Blood RNA MDx Kit provides a rapid, automated procedure to isolate RNA of high quality and purity.
The simple purification procedure begins with a centrifugation step to pellet the contents of each PAXgene Blood RNA Tube. Proteinase K and an optimized incubation buffer are added to digest proteins. The tubes are then transferred to the BioRobot MDx or BioRobot Universal System. After setup of buffers and plasticware, guided by the QIAsoft Operating System, the BioRobot workstation carries out the fully automated RNA purification procedure (see Automated PAXgene RNA MDx purification procedure). Following purification, a final heat treatment of the eluate enhances performance in downstream applications. Total processing time is 3–4 hours, with a maximum of 25–30 minutes of hands-on time. Protocols are available for batches of 48 or 96 samples per run.
The purified RNA 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
|Typical total RNA Yields from PAXgene Blood RNA Tubes processed with BioRobot instruments and the PAXgene Blood RNA MDx Kit||80 KB|
|Certificate of Analysis||Link|
|MSDS Shaker-Adapter, 24-tube, PAXgene||Link|
|MSDS PAXgene 96 Incubator Block||Link|
|MSDS PAXgene Blood RNA MDx Kit||Link|
PAXgene Blood RNA MDx Kit
1. Can the PAXgene Blood RNA MDx 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. Can the PAXgene Blood RNA MDx Kit be used with both the BioRobot Universal System and the BioRobot MDx?
Yes. The PAXgene Blood RNA MDx Kit is designed to be used with both robotic systems.
3. What is the PAXgene 96 filter plate?
The PAXgene 96 filter plate is a special device that filters contaminants and generates a cleared lysate.
4. How many samples can be processed with the PAXgene Blood RNA MDx Kit?
The kit can be used for 384 RNA isolations.
5. What batch sizes can be selected?
Batch sizes of 48 or 96 samples are possible. If other batch sizes are required, please contact QIAGEN technical service (www.qiagen.com).
6. If less than 48/96 samples are being processed, is it possible to leave positions empty?
In general, batch sizes other than 48/96 are not recommended because the vacuum profile can be affected. During a limited number of test runs with 48 or 96 sample batches, up to 8 samples were water blanks instead of real samples. In these runs minimal effects were observed with respect to yield and purity of the eluates.
7. Is there a critical step during upfront sample handling?
Yes. After removing the supernatant by decanting from the PAXgene Blood RNA Tubes, it is sufficient to dab the rim of the tube 5–10 times with a clean paper towel. Excess drying (such as placing the tubes upside down in a rack or wiping the inner walls of the tube) is not recommended and can have negative effects on the automated procedure.
|Product||Catalog Nr.||Price (USD)|
PAXgene Blood RNA MDx Kit (4)For 4 x 96 RNA preps on the BioRobot MDx workstation: 4 PAXgene 96 RNA Plates, 4 PAXgene 96 Filter Plates, Buffers (wash buffers are labeled with bar codes), Proteinase K, RNase-Free DNase Set, Plasticware, Collection Vessels. To be used with PAXgene Blood RNA Tubes.
PAXgene 96 Incubator BlockBlock for denaturation of eluates in PAXgene 96 procedures.
Shaker-Adapter, 24-tube, PAXgeneAdapter for PAXgene Blood RNA Tubes on the shaker unit of the BioRobot Universal System.