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.
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.
The PAXgene Blood RNA procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and resuspended, followed by manual RNA purification.
The PAXgene Blood RNA procedure begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed and resuspended, followed by automated RNA purification.
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).
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).
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).
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).
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).
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).
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).
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.