GLOBINclear™-Mouse/Rat Whole Blood Globin Reduction Kits
Mouse RiboPure™-Blood RNA Isolation Kit
Improved Gene Expression Profiling With Mouse Blood Samples

Expression profiling of blood by microarrays has been historically difficult due to the heterogeneous cellular nature of blood samples and the extremely high concentration of globin transcript present in whole blood total RNA. The Mouse RiboPure™-Blood RNA isolation Kit and GLOBINclear™ Mouse/Rat Globin mRNA depletion technology were used to determine their potential benefits for gene expression profiling. Resulting data showed increased sensitivity and reproducibility on Affymetrix GeneChip® microarrays with globin reduction compared to untreated samples.

RNA Isolation and Globin mRNA Depletion
Total RNA was isolated from two donor C57B6 female mice using Ambion’s Mouse RiboPure™-Blood Kit. RNA from each donor mouse (3.2 µg) was processed with the GLOBINclear™-Mouse/Rat Kit in triplicate. The GLOBINclear Kit employs a novel, subtractive hybridization technology to remove >95% of the α- and β-globin mRNA from whole blood total RNA. When used for expression profiling of human samples, the GLOBINclear Kit processed blood RNA has been shown to increase sensitivity and reproducibility relative to untreated whole blood RNA [1]. Next, 0.9 µg of each GLOBINclear Kit sample was amplified and labeled for use on Affymetrix GeneChip microarrays using the MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit. Untreated whole blood total RNA (0.9 µg) from each donor was also amplified in triplicate. Figure 1 shows representative Agilent® 2100 bioanalyzer traces of amplified RNA (aRNA) before and after GLOBINclear Kit treatment. As can be readily seen in the figure, aRNA from untreated whole blood RNA shows large peaks at ~600 nt, which represent the contribution of globin mRNA to the amplified material. After GLOBINclear Kit processing, these “contaminating” peaks nearly disappear, indicating efficient globin mRNA depletion.

Figure 1. Biotinylated aRNA Trace Derived from GLOBINclear™ Kit RNA or Untreated Whole Blood RNA. aRNA amplified with the MessageAmp™ II-Biotin Enhanced Kit (Ambion Cat #AM1791) was examined on the Agilent® 2100 bioanalyzer. Traces from 1:20 dilution of one representative whole blood aRNA sample and duplicate non-diluted GLOBINclear Kit samples were overlaid to observe the effects of globin mRNA depletion on aRNA profile. The whole blood aRNA is diluted to 1:20 so that the peak from the globin message will be on the same scale as the GLOBINclear aRNA samples. NGC=no GLOBINclear treatment, GC=GLOBINclear Kit-treated sample.

Microarray Analysis
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.

Figure 3. 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.

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