Although whole blood is a readily available source for biological sampling, RNA extraction from blood is often difficult. White blood cells—the most valuable RNA-containing cells—are outnumbered by red cells approximately 1000:1. In addition, once blood is collected, expression levels are threatened by a variety of ex vivo changes, including degradation by potent RNases. Ambion's RiboPure-Blood Kit relies on RNAlater for secure RNA preservation and uses a straightforward RNA isolation method that delivers high yields of quality RNA.
Microarray studies were performed to compare the benefits of RiboPure-Blood Kit–RNAlater with the market leader's system for isolating blood RNA. Whether the samples were processed immediately or stored at room temperature for three days, RNA yields from blood stored in RNAlater were nearly twice that of samples processed with the competitor's system (Figure 1), and the RNA preparations were of comparable quality. Total RNA samples (1 µg) were amplified using Ambion's MessageAmp™ II Kit and hybridized to Affymetrix® Human Focus Microarrays. Scatter plot analyses of the normalized data (average of day 0 and day 3 replicates) revealed that RNAlater was superior to the other manufacturer's lysis solution in preserving the original expression profile (Figure 2).
Figure 2. RNAlater® is Superior to the Market Leader's System in Stabilizing the Expression Profile of Human Whole Blood. Biological replicates of samples processed with RiboPure™-Blood (Ambion) (A) and the market leader's system (B) from day 0 and 3 were compared using Affymetrix® Human Focus Arrays with 10 µg aRNA input without globin reduction. Plots were constructed from signal-normalized data.
Additional array metrics supported this finding (Figure 3). For example, the GAPDH and ß-actin 3'/5' ratios for RiboPure-Blood samples were closer to the ideal value of 1.0. Also, the Percent Present Calls were unchanged after three days of room temperature storage when RNAlater was used. In contrast, over 3% of the present calls—nearly 300 genes—were lost when the other system was used to stabilize blood over the same period. Lastly, RiboPure-Blood enabled a more accurate mRNA profile than the market leader's system. The profiles were dominated by globin mRNA from abundant reticulocytes, and the lower Percent Present Calls at day 0 (RiboPure-Blood compared to market leader's system) were largely due to more accurate representation of globin RNA in RiboPure-Blood samples (data not shown). Ambion's GLOBINclear™ Kit is an ideal companion to RiboPure-Blood to minimize effects of globin mRNA in human blood.
Figure 3. Array Metrics of RNA Isolated from RiboPure™-Blood and the Market Leader's System. A total of 1 µg of RNA purified from both day 0 and day 3 biological replicates were amplified with MessageAmp™ II (Ambion) and hybridized to an Affymetrix® Human Focus Array for analysis.
Figure 4 further emphasizes differences in expression stabilization between RNAlater and the other manufacturer's lysis solution. For both methods, the expression level of every gene on the array was computed as a ratio of the day 0 value to the day 3 value. After the ratios were ordered from high to low for each gene, the ratios for genes in the samples prepared using the market leader's system were plotted against the corresponding genes from RNAlater-preserved samples. As shown in panel A, the day 3 gene expression in the other manufacturer's samples were 0.38 to 2.89 times that of day 0 expression levels. Stabilization in RNAlater spanned a much smaller range (0.64- to 1.62-fold), highlighting hundreds of genes—approximately 10% of the array—that were well conserved in RNAlater, but not with the market leader's protocol. The converse, however, was not true: deviations from 1.0 in the RNAlater gene expression set were lost within the "noise" of the comparative instability of the profile obtained with the other manufacturer's system (Panel B).
RiboPure-Blood Kit with RNAlater offers not only the highest RNA yields, but also significant improvements in mRNA stabilization compared to other commercial methods. The preservation benefits of RNAlater are expected to be particularly valuable in safeguarding blood expression profiles when sample transport is required.
Gary Latham, Jon Kemppainen, Juanita Gonzales, Marianna Goldrick • Ambion, Inc.