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Isolate It All: siRNA miRNA ,,, Total RNA Native Protein

ring sample homogenization, total RNA was isolated from four different mouse tissues with the mirVana PARIS Kit or the mirVana miRNA Isolation Kit. With the latter kit, tissue samples are homogenized directly in a solution containing a chaotropic denaturant, which reduces the opportunities for RNA degradation.

Comparison of the purified RNA on a denaturing gel showed no significant RNA degradation with either kit (Figure 5A). Most importantly, analysis of small RNA expression profiles showed no difference between the two procedures. let-7 microRNA was more abundant in brain than kidney, liver, or thymus, whereas both 5S and 5.8S rRNA were detected at the same level in all 4 tissues. As previously reported [3, 4], miR-124 was expressed only in brain. Together, these results show that homogenization in Cell Disruption Buffer prior to addition of chaotropic denaturant does not significantly affect RNA quality. The advantage is that this lysate contains native protein that can be used directly in a host of downstream applications [2, 5], such as two-dimensional gel electrophoresis (Figure 5B).

Figure 5. Isolation of Total RNA and Protein from Different Tissues. Total RNA and protein were isolated from 4 different mouse tissues (~50 mg) with the mirVana miRNA Isolation Kit (RNA only) or the mirVana PARIS Kit. (A) Small RNA analysis. One g of total RNA was analyzed by denaturing gel and Northern blot as described in Figures 2 and 3.(B) Two-dimensional gel analysis. About 125 g of total protein from mouse brain was resolved using a pH 4-7 IPG gel followed by a 8-1
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