Isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, resuspended, and incubated in optimized buffers containing Proteinase K to digest proteins. A second centrifugation step is carried out to remove residual cell debris, and the supernatant is transferred to a fresh microcentrifuge tube. Ethanol is added to adjust binding conditions, and the lysate is applied to a PAXgene RNA spin column. During a brief centrifugation, RNA is selectively bound to the silica-gel membrane of the spin column as contaminants pass through. Remaining contaminants are removed in three wash steps, and pure RNA is then eluted in Buffer BR5. Generally, DNase digestion is not required for RNA purified with the PAXgene Blood RNA Kit, since PAXgene silica-gel–membrane technology efficiently removes most of the DNA without DNase treatment. However, further DNA removal is recommended for RNA applications that are sensitive to DNA contamination. In these cases, residual DNA can be removed either by using the RNase-Free DNase Set for on-column DNase digestion, or by treating the eluted RNA with DNase.
Label
biotin
Label protocol
Biotin-labeled cRNA were prepared according to the standard Affymetrix protocol from 15 ug total RNA.
Hybridization protocol
Following fragmentation, the cRNA was hybridized for 16 hr at 45C on GeneChip Human Genome U133 Plus 2.0 array. Equilibrate probe array to room temperature immediately before use. Then, heat the hybridization cocktail to 99°C for 5 minutes in heatblock. Next, wet the array by filling it through one of the septa with appropriate volume of 1X MES Hybridization Buffer using a micropipettor and appropriate tips. Last, incubate the probe array for 10 minutes at 45°C in the hybridization oven with rotation.
Scan protocol
The Probe Array is scanned using the Affymetrix System. The scanner is controlled by the GeneChip software. The probe array is scanned after the wash protocols are complete
Description
discovery
Data processing
Quality of arrays was assessed by visual inspection of boxplots, RLE, NUSE and M/A-plots that were produced with the AffyPLM-package (Bolstad et al., 2005) in Bioconductor. The 40 samples were combined with 253 additional Microarray samples from the larger Biomarkers in Transplantation study, and all 293 samples together were background adjusted, normalized and probe information was summarized into probe-sets using the Robust Multi-Array Average (RMA)-procedure from the RefPlus package in Bioconductor (Harbron, Chang, & South, 2007). References: Bolstad, B., Collin, F., Brettschneider, J., Simpson, K., Cope, L., Irizarry, R., & Speed, T. P. (2005). Quality assessment of Affymetrix GeneChip data. Bioinformatics and computational biology solutions using R and bioconductor, 33 47. AND Harbron, C., Chang, K.-M., & South, M. C. (2007). RefPlus: an R package extending the RMA Algorithm. Bioinformatics, 23(18), 2493 2494
