PAXgene Blood RNA Kit产品性能
PAX基因血rna管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在18-25°C：FOS的稳定性“和" RNA在18-25°C：IL1B的稳定性“)，或在2-8°C条件下稳定长达5天(参见" RNA在2-8°C：FOS的稳定性“和" RNA在2-8°C：IL1B的稳定性“，和表格)，高度适合于临床诊断检测.
Reproducible and repeatable RNA purification.
Quadruplicate blood samples from 14 donors were manually processed by each of 3 technicians (A, B, C). Three sets of equipment were used, and all samples prepared by a single technician were processed using the same equipment. [A] Means and standard deviations of RNA yield per replicate samples from the same donors and different technicians are shown. [B] Twelve replicate blood samples from each of 14 donors were processed by the 3 different technicians. Means and standard deviations of RNA yield per samples from the same donors and all technicians are presented. For all RNA samples, A260/A280 ratios ranged from 1.8 to 2.2.
Repeatability and reproducibility of RNA yield.
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. [A] RNA yield and standard deviation for every operator–lot combination. Quadruplicate blood samples from 10 donor pools were processed by 3 different operators (A, B, C) with each of 3 kit lots (1, 2, 3). The mean yields (columns) and standard deviations (error bars) per quadruplicate sample from the same donor pool for different operator and different kit lot are presented. [B] CV of RNA yield per donor pool for all operator–lot combinations (A, B, C; 1, 2, 3) as calculated from the mean yield and standard deviation of the yield shown in part A.
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 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).
Reproducibility between kit lots.
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).
显示的是3名实验员使用3批试剂盒自动化处理2 88个样本的产量.由于使用的是混合血液样本而非单个的PAX基因血RNA管，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。
RNA yield and purity — automated processing.
Blood samples from 48 different donors were collected in PAXgene Blood RNA Tubes (6 tubes per donor, 288 tubes in total). The contents of the tubes from 6 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 6-donor-pool were processed by 3 different operators (A, B, C). Each operator used 3 different lots (1, 2, 3) of the PAXgene Blood RNA Kit for automated extraction and processed quadruplicate samples from each of the 8 donor pools. RNA yields, A260/A280 values, and genomic DNA amounts (w/w) of all individual samples are shown for every operator–lot combination.
当使用多至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β-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PAX基因血RNA系统的自动流程制备的RNA纯度高，没有RT-PCR抑制(见上文)A260/A280“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.
RNA yield and purity — automated processing.
Blood samples from 48 different donors were collected in PAXgene Blood RNA Tubes (6 tubes per donor, 288 tubes in total). The contents of the tubes from 6 donors were pooled and subsequently realiquoted into 36 samples. These 36 samples per 6-donor-pool were processed by 3 different operators (A, B, C). Each operator used 3 different lots (1, 2, 3) of the PAXgene Blood RNA Kit for automated extraction and processed quadruplicate samples from each of the 8 donor pools. RNA yields, A260/A280 values, and genomic DNA amounts (w/w) of all individual samples are shown for every operator–lot combination.
所示，使用PAX基因血RNA系统自动纯化RNA的流程提供高度可重复的RT-PCR结果，适用于临床诊断检测。
Reproducibility between automated and manual protocols.
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).
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