Biotin labeled aRNA derived from both donor mice, with and without GLOBINclear Kit treatment, was hybridized to Affymetrix Mouse 430A 2.0 GeneChips to assess the sensitivity and reproducibility of the methods (2 donors x 3 replicates x 2 treatments = 12 arrays). Increased sensitivity resulting from different RNA processing protocols can be evaluated in several ways: number of genes called above background (Present Calls), scaling factor, and signal intensity. As can be seen in Figure 2, Present Calls, scale factor, and mean signal all indicate that GLOBINclear Kit processing results in increased signal-to-noise ratios, and, hence, increased sensitivity. An average of 4753 and 5965 additional genes was called Present after globin depletion for Donor 37 and Donor 45, respectively. This represents an average increase of 119% genes that were called Present. Scale factor also decreased substantially with GLOBINclear Kit treatment, confirming the method’s positive effect on global signal. Scale factor is an indirect measure of signal, with lower values indicating higher array signal. Lastly, the actual mean signal increased by nearly 30% caused by the GLOBINclear Kit treatment. This last measure can also be observed in the signal distribution histograms in Figure 3. For both donors, the signal distributions shifted to the right (higher) and tailed off more gradually after globin mRNA depletion (also see the signal standard deviation in Figure 2). This indicates an increase in average signal and greater sensitivity throughout the distribution. Higher signal, especially for weakly expressed genes, results in better estimates.
Signal Distribution Histograms for Affymetrix Mouse 430A 2.0 GeneChips®. Log2 signal distributions are plotted for all four treatment/donor conditions (A–D). Each distribution represents the average of triplicate technical replicates. Signal was estimated by RMA without quantile normalization as in Figure 2. The box plots shown above each histogram indicates the mean and median values, the interquartile range (sides of box) and the upper and lower 0.5%, 2.5%, and 10% quantiles. JMP Statistical Analysis Software was used to build the plots.
To summarize, this study demonstrated the beneficial effects on sensitivity and reproducibility that can be achieved by the Mouse RiboPure -Blood RNA Isolation Kit and GLOBINclear-Mouse/Rat Globin mRNA Depletion technology. These methods introduce new opportunities in the field of expression profiling of difficult sample types, such as mouse and rat blood. Scientific Contributors
Penn Whitley, Juanita Gonzales, Marianna Goldrick • Ambion Inc.