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Isolate Total RNA and Protein From the Same Sample

//www.ambion.com/catalog/CatNum.php?">Vana miRNA Detection Kit [2, 3], miR-16 miRNA could barely be detected in 1 g of total RNA isolated from HeLa cells in this way. In contrast, RNA isolated using both the mirVana miRNA Isolation Kit and mirVana PARIS Kit procedures included these small RNA species.

Figure 1. Differential Recovery of Small RNAs. Total RNA was isolated from 1x106 HeLa cells with the indicated kit as per protocol. One g of total RNA was resolved on a 15% denaturing acrylamide gel and stained with EtBr (top) or analyzed by solution hybridization assay with the mirVana miRNA Detection Kit (Ambion) and a probe specific for miR-16 prepared by in vitro transcription with the mirVana Probe Construction Kit (Ambion). Gel was exposed for 6 (middle) or 24 (bottom) hours at -80C


Measuring Reproducibility

Variation in protein, mRNA, and/or small RNA recovery can be an issue if precise quantitation is required in downstream experiments (e.g. RNAi experiments, miRNA profiling). Therefore, reproducible, quantitative recovery of RNA and protein is critical. To determine the reproducibility of the mirVana PARIS procedure, both RNA and protein were isolated from replicates of two types of biological samples, and various quantitative and qualitative parameters were measured. The experimental samples tested were human cell lines assayed 2 days after plating (12 replicates, Figure 2), and mouse liver tissue kept frozen for 2 months at -80C prior to assay (6 replicates, Figure 3). For each experiment, total RNA and protein yields were determined, and samples were assessed for effi
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