PAXgene Blood RNA Tubes are intended for the collection of whole blood and stabilization of intracellular RNA for up to 3 days at 18–25°C (see figures "RNA stability at 18–25°C: FOS" and "RNA stability at 18–25°C: IL1B") or up to 5 days at 2–8°C (see figures " RNA stability at 2–8°C: FOS" and " RNA stability at 2–8°C: IL1B"). Currently available data shows stabilization of cellular RNA for at least 96 months at –20°C or –70°C.
Total RNA purified using the PAXgene Blood RNA System is highly pure, with A260/A280 values between 1.8 and 2.2 and ≤1.0% (w/w) genomic DNA. At least 95% of samples show no inhibition in RT-PCR, when using up to 30% of the eluate.
Average sample preparation time (based on data from 12 sample preps) is approximately 90 minutes, with only 40 minutes of hands-on time. RNA yields from 2.5 ml healthy human whole blood are ≥3 µg for ≥95% of the samples processed. Since yields are highly donor-dependent, individual yields may vary. For individual donors, the PAXgene Blood RNA System provides highly reproducible and repeatable yields (see figures " Reproducible and repeatable RNA purification" and " Repeatability and reproducibility of RNA yield") and reproducible and repeatable RT-PCR (see figures " Reproducibility between users" and " Reproducibility between kit lots", and table), making it highly robust for clinical diagnostic tests.
RNA yields from 2.5 ml healthy human whole blood are ≥3 μg for ≥95% of the samples processed. The figure " RNA yield and purity — automated processing" indicates the RNA yields from a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators. As pooled blood samples instead of individual PAXgene Blood RNA Tubes were used for these studies, the results do not reflect the RNA yield expected from single samples of individual blood draws. Since yields are highly donor-dependent, individual yields may vary.
At least 95% of samples show no inhibition in RT-PCR, when using up to 30% of the eluate. Using the automated protocol, cross contamination between samples is undetectable, as measured by quantitative, real-time RT-PCR of sequences of the betaglobin and FOS transcripts in RNA-negative samples (water) paired with RNA-positive samples (human whole blood) in the same run.
RNA purified with the PAXgene Blood RNA System and the automated protocol is highly pure, as shown by lack of RT-PCR inhibition (see above) and A260/A280 values between 1.8 and 2.2. Genomic DNA is present at ≤1% (w/w) in ≥95% of all samples, as measured by quantitative, real-time PCR of a sequence of the beta-actin gene. The figure " RNA yield and purity — automated processing" shows the A260/A280 values and relative genomic DNA of a total of 288 samples prepared using the automated protocol with 3 kit lots by 3 operators.
The automated protocol of RNA purification using the PAXgene Blood RNA System provides highly reproducible and repeatable RT-PCR results, as shown in the figure " Reproducibility between automated and manual protocols", making it highly robust for clinical diagnostic tests.
| Comparison of data | Mean | ± SD | Mean | ± SD |
| | (ΔΔCT) | (ΔΔCT) | (ΔΔCT) | (ΔΔCT) |
| Reproducibility within each user and between all lots |
| All users, lot 1 – lot 1 | 0.00 | 0.00 | 0.00 | 0.00 |
| All users, lot 1 – lot 2 | –0.03 | 0.48 | –0.07 | 0.66 |
| All users, lot 1 – lot 3 | –0.21 | 0.52 | 0.11 | 0.71 |
| Reproducibility within each lot and between all users |
| All lots, user A – user A | 0.00 | 0.00 | 0.00 | 0.00 |
| All lots, user A – user B | –0.46 | 0.44 | –0.06 | 0.69 |
| All lots, user A – user C | –0.31 | 0.60 | –0.15 | 0.71 |
User: Technician, performed the study.
Lot: Number of kit lot used in this study.
SD: Standard deviation.
Mean ΔΔCT values (N = 120) and standard deviations are shown for the data presented in the figures " Reproducibility between users" and " Reproducibility between kit lots".
