PAXgene Blood RNA Kit產(chǎn)品性能
PAX基因血rna管適用于采集全血并穩(wěn)定細(xì)胞內(nèi)rna，在18-25°C條件下可穩(wěn)定長(zhǎng)達(dá)3天(參見(jiàn)“ RNA在18-25°C：FOS的穩(wěn)定性“和” RNA在18-25°C：IL1B的穩(wěn)定性“)，或在2-8°C條件下穩(wěn)定長(zhǎng)達(dá)5天(參見(jiàn)” RNA在2-8°C：FOS的穩(wěn)定性“和” RNA在2-8°C：IL1B的穩(wěn)定性“，和表格)，高度適合于臨床診斷檢測(cè).
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).
顯示的是3名實(shí)驗(yàn)員使用3批試劑盒自動(dòng)化處理2 88個(gè)樣本的產(chǎn)量.由于使用的是混合血液樣本而非單個(gè)的PAX基因血RNA管，實(shí)驗(yàn)結(jié)果不能反映單一個(gè)體血液樣本的RNA預(yù)期產(chǎn)量.由于產(chǎn)量高度依賴于供體，單個(gè)產(chǎn)量會(huì)有差別。
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.
當(dāng)使用多至30%的洗脫液時(shí)，至少95%的樣品不會(huì)出現(xiàn)RT PCR抑制.通過(guò)對(duì)RNA陰性樣品(水)中β-球蛋白和fos轉(zhuǎn)錄子序列與RNA陽(yáng)性樣本(人類全血)進(jìn)行定量rt-PCR分析，未發(fā)現(xiàn)使用自動(dòng)化流程處理樣品中有交叉污染.
使用PAX基因血RNA系統(tǒng)的自動(dòng)流程制備的RNA純度高，沒(méi)有RT-PCR抑制(見(jiàn)上文)A260/A280“顯示了3個(gè)實(shí)驗(yàn)員使用3批試劑盒處理288個(gè)樣品的A260/A280值和相對(duì)基因組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系統(tǒng)自動(dòng)純化RNA的流程提供高度可重復(fù)的RT-PCR結(jié)果，適用于臨床診斷檢測(cè)。
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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