Additional File 1.
Quantitative measures of RNA degradation, DegFact and RIN, based on electrophoretic traces shown in Fig. 1. Larger DegFact (scale of 0–100%) and smaller RIN (scale of 1–10) indicate more degradation. 817A: prostate cancer tissue; 817B: matched benign prostate tissue; 957B: benign prostate tissue; UN: unstained; WOUT: stained without RNase inhibitor; W: stained in the presence of RNase inhibitor; YELLOW: degradation can be detected; ORANGE: severe degradation; RED: highest alert, strong degradation. To standardize interpretation of RNA integrity, quantitative measures of RNA degradation based on electropherograms have been developed. With increasing degradation, heights of 18S and 28S peaks gradually decrease and additional 'degradation peak signals' appear in a molecular weight range between small RNAs and the 18S peak . The degradation factor (DegFact, %Dgr/18S) is defined as the ratio of the average degradation peak signal (30–41 seconds) to the 18S peak signal (41–42.5 seconds) multiplied by 100 . The larger the degradation factor, the more degraded the sample. The RNA Integrity Number (RIN) allows for classification of eukaryotic total RNA based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being the most intact . Degradation factors and RINs based on the electropherograms in Figures 1A–1C are listed in Additional file 1. For all three cases, degradation factors were higher and RINs were lower for sections stained in the absence of the RNase inhibitor relative to serial sections that were unstained. Degradation factors were decreased and RINs were increased for tissues stained in the presence compared to the absence of RNase inhibitor, indicating a protective effect against RNA degradation. Thus, the quantitative measures were in accordance with visual interpretations. Interestingly, tissues that appeared similar in terms of RNA integrity when unstained were observed to differ significantly in RNA degradation after staining, especially in the absence of RNase inhibitor. As shown in Additional file 1, the degradation factors were similar for 817A (9.7) and 817B (9.6) when the tissues were unstained; however, the degradation factors for 817A (14.6) and 817B (30.9) were very different after staining without RNase inhibitor. Even in the presence of RNase inhibitor, a significant difference remained between 817A (12.6) and 817B (18.5). Thus, differences in tissue quality that are not apparent before staining can become evident after staining. It can be more informative, therefore, to analyze tissue sections for RNA integrity after fixing, staining, and dehydrating rather than analyzing unstained tissues.
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Kube et al. BMC Molecular Biology 2007 8:25 doi:10.1186/1471-2199-8-25