PAXgene Blood RNA Tubes

分离和纯化稳定在PAXgene Blood RNA Tubes中血液的细胞内RNA

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PAXgene Blood RNA Tube (100), IVD

Cat. No. / ID: 762165

100 PAXgene Blood RNA Tubes. This product is sold by BD. Clicking inquire will take you to BD Customer Care.

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These products are not available in all countries; please inquire.

PAXgene Blood RNA Tubes适用于体外诊断。

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特点

符合欧盟体外诊断98/79/EC指令
用于采样、稳定及纯化的整合系统
RNA在18–25°C条件下可稳定多达3天
在–20°C或–70°C条件下至少稳定60个月
分析前标准化的样本处理过程

产品详情

PAXgene Blood RNA体系包括用于血液采集、稳定和运输的PAXgene Blood RNA Tubes（由BD提供，货号762165），以及基于硅胶膜技术、离心柱法分离纯化的PAXgene Blood RNA Kit。纯化可使用离心机进行手工操作，也可在QIAcube全自动核酸纯化仪上自动运行。该产品表现卓越，确保高度可靠的RNA纯化。

绩效

PAXgene Blood RNA Tubes适用于采集全血并稳定细胞内RNA，在18–25°C条件下可稳定长达3天（参见"RNA stability at 18–25°C: FOS"和"RNA stability at 18–25°C: IL1B"），或在2–8°C条件下稳定长达5天（参见"RNA stability at 2–8°C: FOS"和"RNA stability at 2–8°C: IL1B"）。当前的数据显示，细胞内RNA在–20°C或–70°C条件下可保存至少60个月。

使用PAXgene Blood RNA System纯化的总RNA纯度高，A₂₆₀/A₂₈₀值在1.8和2.2之间，且基因组DNA含量不超过1.0% (w/w)。当使用30%的洗脱液时，至少95%的样品在RT-PCR中没有出现反应抑制的情况。样本制备的平均时间约为90分钟（根据12个样本的制备时间），手工操作只需40分钟。对于95%以上的样本，2.5 ml健康人类全血的RNA产量不低于3 µg。由于产量高度依赖于供体，单次产量可能会有变动。对于单个供体，PAXgene Blood RNA System确保产量可重复性高（参见"Reproducible and repeatable RNA purification"和"Repeatability and reproducibility of RNA yield"）以及RT-PCR的可重复性高（参见"Reproducibility between users"、"Reproducibility between kit lots"和下方表格）。

对于超过95%的样本，从2.5 ml健康人类全血中获得的RNA产量不少于3 μg。图片"RNA yield and purity — automated processing"显示的是3名实验员使用3批试剂盒自动化处理288个样本的RNA产量。由于使用的是混合血液样本而非单个的PAXgene Blood RNA Tubes，实验结果不能反映单一个体血液样本的RNA预期产量。由于产量高度依赖于供体，单个产量会有差别。

当使用30%的洗脱液时，至少95%的样品不会出现RT-PCR抑制。通过对RNA阴性样品（水）中beta-球蛋白和FOS转录子序列与RNA阳性样本（人类全血）进行定量RT-PCR分析，未发现使用自动化流程处理样品中有交叉污染。

使用PAXgene Blood RNA System的自动流程制备的RNA纯度高，没有RT-PCR抑制（见上文），A₂₆₀/A₂₈₀值在1.8 和2.2之间。经real-time PCR分析beta-肌动蛋白的序列发现，超过95%的样品的基因组DNA含量不高于1% (w/w) 。图片"RNA yield and purity — automated processing"显示了3个实验员使用3批试剂盒处理288个样品的A₂₆₀/A₂₈₀值和相对基因组DNA含量。

如图"Reproducibility between automated and manual protocols"所示，使用PAXgene Blood RNA System自动纯化RNA的流程提供高度可重复的RT-PCR结果。

RT-PCR数据总结
检测系统	FOS/18S rRNA assay		IL1B/18S rRNA assay
数据对比	平均值	± SD	平均值	± SD
	(ΔΔC_T)	(ΔΔC_T)	(ΔΔC_T)	(ΔΔC_T)
同一使用者使用不同批试剂盒的可重复性
所有使用者，第一批–第一批	0.00	0.00	0.00	0.00
所有使用者，第一批–第二批	–0.03	0.48	–0.07	0.66
所有使用者，第一批–第三批	–0.21	0.52	0.11	0.71
不同使用者使用同一批试剂盒的可重复性
所有批次，使用者A–使用者A	0.00	0.00	0.00	0.00
所有批次，使用者A–使用者B	–0.46	0.44	–0.06	0.69
所有批次，使用者A–使用者C	–0.31	0.60	–0.15	0.71

原理

PAXgene Blood RNA Kit用于从在PAXgene Blood RNA Tube中采集的2.5 ml人类全血中纯化总RNA，操作简单，可手工操作，也可自动化运行（参见"Manual PAXgene Blood RNA procedure"和"Automated PAXgene Blood RNA procedure"）。

PAXgene Blood RNA Tubes 含有专利申请中的特定试剂，基于RNA固定的专利技术（US Patents 6,602,718 和6,617,170）。这些试剂成分可保护RNA分子，避免其被RNA酶降解，并将基因表达体内变化的改变降至最小。PAXgene Blood RNA Tubes适用于采集全血并稳定细胞内RNA，在18–25°C条件下可稳定长达3天（参见" RNA stability at 18–25°C: FOS"和" RNA stability at 18–25°C: IL1B"）或在2–8°C条件下稳定长达5天（参见" RNA stability at 2–8°C: FOS"和" RNA stability at 2–8°C: IL1B"）。当前的数据显示，细胞内RNA在–20°C或–70°C条件下可保存至少60个月。

查看图表

RNA stability at 18–25°C: FOS.

RNA stability at 18–25°C: IL1B.

RNA stability at 2–8°C: FOS.

RNA stability at 2–8°C: IL1B.

程序

PAXgene Blood RNA纯化操作流程简单，可手动或自动进行（参见"Manual PAXgene Blood RNA procedure"和"Automated PAXgene Blood RNA procedure"）。

PAXgene Blood RNA纯化手动流程

将重悬的沉淀和蛋白酶K一起溶解在经优化的缓冲液中孵育，进行蛋白质酵解。然后应用PAXgene Shredder离心柱再次离心，匀浆细胞裂解液，并去除剩余的细胞残骸。将流过部分的上清液转移到干净的微型离心管中，加入乙醇调节结合条件，将裂解液装入一个PAXgene RNA离心柱中。快速离心后，RNA会选择性地结合到PAXgene硅胶膜上，杂质则流过。剩余的杂质通过几次高效洗涤去除。在第一次和第二次洗涤步骤之间，使用DNA酶处理膜，以去除微量残余的DNA。洗涤之后，使用洗脱缓冲液洗脱RNA并进行热变性。

PAXgene Blood RNA纯化自动流程

使用QIAcube全自动核酸纯化仪制备样品的流程与手动法相同，您可继续使用PAXgene Blood RNA Kit纯化高品质RNA。

自动化的RNA纯化流程由两个部分组成，"PAXgene Blood RNA Part A"和"PAXgene Blood RNA Part B"，在两个部分之间需要短暂的手工操作。

将经离心、洗涤、重悬的核酸沉淀从PAXgene Blood RNA Tube转移到离心管中，将离心放到QIAcube全自动核酸纯化仪工作台的恒温震荡器中。实验员选择并启动菜单中的"PAXgene Blood RNA Part A"。QIAcube全自动核酸纯化仪会自动运行，直到将RNA洗脱至洗脱缓冲液中。实验员将含有已纯化RNA的离心管转移到QIAcube全自动核酸纯化仪的恒温震荡器中，选择并启动菜单中的"PAXgene Blood RNA Part B"，QIAcube全自动核酸纯化仪会执行热变性。

样本平均制备时间是125分钟（基于制备12个样本的时间），手工操作时间只需约20分钟。

查看图表

应用

该试剂盒与PAXgene Blood RNA Tubes共同使用，可从全血中分离纯化细胞内RNA进行RT‑PCR。

辅助数据和图表

RNA stability at 18–25°C: FOS.

Blood was drawn from ten apparently healthy consented 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 C_T).

Specifications

Features	Specifications
Tube size	16 x 100 mm
Draw volume	2.5 ml
Additive/concentration	Additive 6.9 ml
Closure type/color	BD Hemogard™ safety closure with transparent shield and red stopper
Label type	Paper
Quantity	8.5 ml

资源

For collection, transport, and storage of whole blood and stabilization and purification of intracellular RNA

For molecular diagnostic testing

Guenther et al., AMP 2005

Guenther et al., AACR 2007

Guenter et al., ISBER 2009

Guenther et al., CHI Genomic Sample Prep 2009

Typical total RNA yields from PAXgene Blood RNA Tubes processed with the PAXgene Blood RNA Kit

In situ stability of RNA in blood specimens stored for 11 years (132 months) at –20°C and –70°C in PAXgene Blood RNA Tubes

FAQ

RNA yields isolated from 2.5 ml healthy, human, whole blood (4.8 x 10⁶ –1.1 x 10⁷ leukocytes/ml) are specified to ≥3 µg for >95% of the samples processed (see Technical Note for the QIAsymphony PAXgene Blood RNA System ). Since yields are highly donor-dependent, individual yields may vary. Mean yields in the range of 8-10µg per tube or higher.

FAQ ID - 3475

You can find additional information relating to the PreAnalytiX PAXgene products on the PreAnalytiX website .

FAQ ID - 3515

The PAXgene Blood RNA System was developed for intracellular RNA isolation from human blood samples. QIAGEN offers RNA stabilization and isolation systems for animal blood. Please contact QIAGEN technical service (www.qiagen.com) for more information.

FAQ ID - 3461

Typical yields of RNA isolated from 2.5 ml healthy, human, whole blood (4.8 x 10⁶ –1.1 x 10⁷ leukocytes/ml) are ≥3 μg for >95% of the samples processed (Refer to Technical Note) . Since yields are highly donor-dependent, individual yields may vary.

FAQ ID - 3481

Typical yields of RNA isolated from 2.5 ml human whole blood are between 4 and 20 µg. However yield is highly donor-dependent, and in some cases higher or lower yields may be achieved. At least 95% of all samples with a WBC count between 4.8 and 11.0 x 106 cells/ml of blood will yield ≥3 µg of total RNA per tube. To prevent low yields due to underfilling of the tube, see the phlebotomy FAQ and the blood collection demonstration video.

FAQ ID - 3464

A blood collection set is required to be used with the PAXgene Blood RNA Tube to eliminate the possibility of patient contact with the reagent in the tube, due to backflow of blood, when used in accordance with the instructions for use.

FAQ ID - 3459

Like yield, RNA integrity depends on parameters, such as tissue type, period of cold ischemia, fixation time, processing protocol, age, and storage conditions of the PFPE block. For examples of the integrity of RNA in rat tissues under ideal workflow conditions, see Groelz et al., Exp Mol Pathol. 2013 Feb;94(1). For examples of the integrity of RNA from clinical samples, see Viertler et al., J Mol Diagn. 2012 Sep;14(5)

FAQ ID -3046

Currently we have data for storage at –20°C and –70°C for 96 months (In situ stability of RNA in blood specimens stored for 8 years (96 months) at –20°C and –70°C in PAXgene Blood RNA Tubes).

Long term archiving studies are ongoing. See technical note: In Situ Stability of RNA in Blood Samples Stored at –20°C and –70°C in PAXgene Blood RNA Tubes.

FAQ ID - 3463

Yes. You may dispose of the waste product from the PAXgene Blood RNA Tube with a 5% sodium hypochlorite bleach solution, in a 1:9 ratio (1 part bleach : 9 parts tube contents).

FAQ ID - 3469

Possible reasons for blood draws with lower than expected blood volume include: 1) The phlebotomist has not waited enough time for the blood to stop flowing into the tube. 2) The tube has "pushed back" from the needle end attached to the holder and the tube is no longer drawing. 3) Discard tube was not used. For further information see the video Collecting Specimens In PAXgene Blood RNA Tubes and Instructions for Use.

FAQ ID - 3462

If used according to the instructions for use, there is no possibility of backflow during phlebotomy, and thus no possibility of reagent going into the patient’s arm.

FAQ ID - 3460

Yes. Samples have to be incubated at least 2 h at room temperature (18-22°C). Incubation of the PAXgene Blood RNA Tube overnight may increase yields slightly in some cases. Room temperature storage for up to three days has no effect on the RNA profile. During the preparation of the RNA the manual pretreatment consist of a centrifugation and a pellet resuspenion step (see the PAXgene Blood miRNA Handbook ).

FAQ ID - 3479