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RNA Yield, Purity, and Quality
RNA yield, purity and quality are factors that are important for successful gene expression analysis. Typically, yields for total RNA follow the “1/1000th rule”, i.e. one can expect to get about 1 μg of RNA for every mg of tissue. This rule varies with tissue type, e.g. skin yield is much less, but most yields are within a 5-fold level. RNA yield can be measured by looking at the A₂₆₀ reading. A reliable and inexpensive method to look at RNA quality is to run the samples on a polyacrylamide gel.

Case Study
In our study, 250 ng RNA from 1 biological replicate set was combined with 5 μL of Ambion’s Gel Loading Buffer II and concentrated using a Savant SpeedVac® on medium heat to a final volume of 10 μL. Samples were then incubated for 2 minutes at 95°C and immediately placed on ice. Decade™ Marker was prepared according to protocol using Ambion’s mirVana™ Probe & Marker Kit. Samples were run on a polyacrylamide gel (Figure 2 below).

Figure 1 (on Main Page) demonstrates efficient recovery of total and enriched miRNA from tissues of 3 different animals using the mirVana Isolation Kit. About 1 μg of total RNA was recovered in each animal for all tissue types. Since tRNA and other small functional RNAs comprise 5–20% of the total RNA population, the gross recovery of enriched RNA by A₂₆₀ was about a tenth that of total, but the amount of miRNA present was about the same.

Figure 2 (below) illustrates the yield of the varying RNA sizes run on a polyacrylamide gel, where losses in yield are due to losing the high molecular weight RNA species that are embedded in the gel at the origin. Here also the recovery of enriched RNA is about a tenth that of the total RNA.

Figure 2. RNA Yield in Frozen Versus RNAlater® Solution-Treated Samples. The greatly enriched presence of tRNA (~70 nt) is apparent, as equal amounts of RNA were loaded. The RNAlater Solution-treated samples (A) provide equivalent samples in terms of banding patterns when compared to frozen samples (B). The enrichment procedure is not totally size-dependent, but also enriches for some small RNAs preferentially (perhaps due to structural qualities). The mass of large RNAs (trapped in the gel origin) are greatly reduced. The lower molecular weight bands seen in the lung samples are occasionally seen in this sample type and could be degradation products. Each sample was run on a 7 M urea/15% polyacrylamide gel with 1 μL unlabeled Decade™ Markers (MKRS). Prior to sample loading, gels were run at 300 V for 10 min, and the wells were flushed with 1X TBE Buffer. The gel was run at 200 V until exit of the bromophenol blue dye front from the gel. Gels were stained for 30 minutes with a 1:10,000 dilution of SYBR® Gold Dye and photographed using Alphaimager v5.5 software. Sample loading was standardized according to ng RNA loaded.

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