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Performance
The patented additive in PAXgene Blood RNA Tubes protects RNA molecules from degradation by RNases and other enzymes and triggers the complete lysis of all cells in the sample to minimize ex vivo changes in gene expression due to gene induction and downregulation. The result is immediate and long-term stabilization of RNA in specimens, even after being stored for up to 3 days at room temperature (Figure 1 and Figure 2) or up to 5 days refrigerated (Figure 3 and Figure 4). Currently available data show stabilization of intracellular RNA for up to 11 years at –20°C or –70°C (see Technical Notes).
Principle
The collection of whole blood is the first step in many molecular assays used to study intracellular RNA. A major challenge in this type of testing is the instability of intracellular RNA, which rapidly degrades within hours after blood collection. Furthermore, certain transcript levels change after blood collection due to ex vivo gene regulation, such as induction and repression. Both RNA degradation and gene regulation can lead to an under- or overestimate of in vivo gene transcript levels.
The PAXgene Blood RNA Tube contains an additive that stabilizes the in vivo gene expression profile by reducing post-collection RNA degradation and minimizing gene induction and repression. When used in conjunction with one of the PAXgene Blood RNA Kits, the PAXgene Blood RNA Tube provides pure RNA for accurate detection and quantification of gene expression.
Procedure
Whole blood is collected directly into the PAXgene Blood RNA Tube, using standard phlebotomy practice and equipment. The RNA in the specimen is immediately stabilized for storage at room temperature, refrigerated, or frozen. RNA can be purified using one of the PAXgene Blood RNA Kits (choose the right PAXgene Blood RNA Kit). The ease of collection, stabilization of RNA at different temperatures, and elimination of additional processing steps (e.g., centrifugation, storage on ice) standardizes the workflow from collection to purification, reducing variability that can be introduced by different operators (Figure 5).
Applications
When used in conjunction with the PAXgene Blood RNA Kit, the PAXgene Blood RNA Tubes enable purification of intracellular RNA from whole blood for applications in molecular diagnostic testing, such as real-time RT-PCR analysis of specific transcripts.
PAXgene Blood RNA Tubes are intended for in vitro diagnostic testing (IVD) and are to be used in conjunction with the PAXgene Blood RNA Kit.
![Blood was drawn from 10 donors, with duplicate samples, and stored at 18–25°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane–based RNA cleanup. Relative transcript levels of FOS were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (2.34 CT).](/storage/images/Content/Blood/RNA_miRNA/RNA_stability_at_18_25C_FOS.jpg "Figure 1. RNA stability at 18–25°C: FOS.")
![Blood was drawn from 10 donors, with duplicate samples, and stored at 18–25°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane–based RNA cleanup. Relative transcript levels of IL1B were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (1.93 CT).](/storage/images/Content/Blood/RNA_miRNA/RNA_stability_at_18.25_C_IL_18.jpg "Figure 2. RNA stability at 18–25°C: IL1B")
![Blood was drawn from 10 donors, with duplicate samples, and stored at 2–8°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane-based RNA cleanup. Relative transcript levels of FOS were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (2.34 CT).](/storage/images/Content/Blood/RNA_miRNA/RNA_stability_at_2-8_C_FOS.jpg "Figure 3. RNA stability at 2–8°C: FOS.")
![Blood was drawn from 10 donors, with duplicate samples, and stored at 2–8°C for the indicated number of days, followed by total RNA purification. [A] Blood was collected and stored in PAXgene Blood RNA Tubes, and total RNA was purified using the PAXgene Blood RNA Kit. [B] Blood was collected and stored in standard blood collection tubes with EDTA as an anticoagulant, and total RNA was purified using a standard organic-extraction method with silica-membrane-based RNA cleanup. Relative transcript levels of IL1B were determined by real-time, duplex RT-PCR, using 18S rRNA as an internal standard. The values for all samples are plotted, with means and standard deviations of all samples shown. The dashed lines indicate the ±3x total precision of the assay (1.93 CT).](/storage/images/Content/Blood/RNA_miRNA/RNA_stability_at_2-8_C_IL_1B.jpg "Figure 4. RNA stability at 2–8°C: IL1B.")
![Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes (12 tubes per donor, 360 tubes in total). The contents of the tubes from 3 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 3-donor-pool were manually processed by 3 different operators. Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools. Relative transcript levels of [A] FOS and [B] IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. The values for all samples are plotted, relative to the values for user 1 (10 donor pools x 3 kit lots x 4 replicates = 120 data sets for each gene), with means (red lines) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS, 2.34 CT; IL1B, 1.93 CT).](/storage/images/Content/Blood/RNA_miRNA/Reproducibility_between_users.jpg "Figure 5. Reproducibility between users.")
![Blood samples from 30 different donors were collected in PAXgene Blood RNA Tubes (12 tubes per donor, 360 tubes in total). The contents of the tubes from 3 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 3-donor-pool were manually processed by 3 different operators. Each operator used 3 different PAXgene Blood RNA Kit lots for the extraction and processed quadruplicate samples from each of the 10 donor pools. Relative transcript levels of [A] FOS and [B] IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. The values for all samples are plotted, relative to the values for kit lot 1 (10 donor pools x 3 users x 4 replicates = 120 data sets for each gene), with means (red lines) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS, 2.34 CT; IL1B, 1.93 CT).](/storage/images/Content/Blood/RNA_miRNA/Reproducibility_between_kit_lots.jpg "Figure 12. Reproducibility between kit lots.")
![RNA was purified by 3 different operators (A, B, C) using 3 different lots (1, 2, 3) of the PAXgene Blood RNA Kit using the automated protocol in the experiment described in the figure "RNA yield and purity — automated processing". In parallel, RNA was purified from the corresponding replicate tubes using the manual protocol. Relative transcript levels of [A] FOS and [B] IL1B were determined by real-time, duplex RT-PCR using 18S rRNA as an internal standard. Possible differences of transcript levels between RNA prepared from paired blood samples using both extraction protocols (automated and manual protocol) were calculated by the ΔΔCT method. Individual ΔΔCT values for all sample pairs (4 replicates x 8 donor pools x 3 kit lots x 3 operators = 288 pairs for each gene) are plotted as single dots with means (larger dots) and standard deviations (black bars) for all samples shown. The dashed lines indicate the ±3x total precision of the assays (FOS: 2.34 CT; IL1B, 1.93 CT).](/storage/images/Content/Blood/RNA_miRNA/Reproducibility_between_automated_and_manual_protocols.jpg "Figure 13. Reproducibility between automated and manual protocols.")
