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Isolate Total RNA and Protein From the Same Sample

cient recovery of long RNA species (real time RT-PCR), small RNA (gel or Northern blot), and protein (Western blot). The results showed that high quality total RNA and protein were efficiently recovered from both samples types. No apparent degradation was observed by gel analysis or Western blot. The mirVana PARIS isolation procedure was also extremely reproducible as each independent replicate contained the same relative proportion of long RNA (18S rRNA, GAPDH, and/or Cyclin D1 mRNA), small RNA (5S rRNA), and protein (GAPDH). Quantitative analysis by real time PCR showed variations of less than 10% (Figure 4).

Figure 2. Analysis of 12 Human Cultured Cell Replicates. HeLa cells were plated at 200,000 cells per well in 6 well plates. Total RNA and protein were isolated 2 days after plating with the mirVana PARIS Kit. One g of total RNA was analyzed on a 15% denaturing polyacryl-amide gel stained with EtBr (left top). Ten g of total protein was resolved on a 10% SDS polyacrylamide gel and analyzed by Western blot with an anti-GAPDH antibody (Ambion) (left bottom). Real time PCR reactions were performed in duplicate using the indicated gene specific primers in combination with SYBR green technology (right).

Figure 3. Analysis of 6 Mouse Liver Replicates. Total RNA and protein were isolated with the mirVana PARIS Kit from 50-80 mg pieces of liver dissected from 6 different mice and kept frozen at -80C for 2 months. One g of total RNA was analyzed on a 1.2% denaturing glyoxal agarose gel stained with EtBr or resolved on a 15% denaturing polyacrylamide gel and analyzed by Northern blot with a 5S rRNA pr
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