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Performance
The PAXgene 96 Blood RNA Kit allows high-throughput isolation of total RNA from human whole blood. The robust PAXgene 96 Blood RNA procedure is highly reproducible and typically yields are between 4 and 20 µg of RNA from 2.5 ml human whole blood with minimal risk of sample-to-sample cross contamination. The high-throughput procedure is simple, fast, and efficient, and processing 96 samples in parallel provides significant time savings.
Principle
Copy numbers of individual RNA species in blood can change significantly during storage or transport at ambient temperature, making reliable studies of gene expression impossible. Both stabilization of RNA molecules after collection and an efficient RNA purification protocol are needed to maximize insights into expression profiles of whole blood. Based on proven silica-membrane technology in a high-throughput, 96-well plate format, and when combined with PAXgene Blood RNA Tubes, the PAXgene 96 Blood RNA Kit provides the chemistry and rapid procedure to isolate RNA of high quality and purity.
Procedure
RNA isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, and proteinase K is added to digest proteins. Alcohol is added to adjust binding conditions. Lysates are applied to the PAXgene 96 Filter Plate and centrifuged to remove cell debris. The lysate is then applied to a PAXgene 96 RNA Plate (Figure 1). Residual DNA is removed through a DNase I digestion. Remaining contaminants are removed in three efficient wash steps before pure RNA is eluted.
Applications
The purified RNA is ready for use in a wide range of downstream applications, including:
- cDNA synthesis
- Gene expression arrays
- End-point RT-PCR
- Quantitative RT-PCR, including TaqMan technology
- Next-generation sequencing
