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

, resulting in rapid inactivation of cellular ribonucleases. After a rapid extraction with Acid-Phenol:Chloroform (provided with the kit), total RNA was then purified from the mixture using an RNA-binding glass fiber filter (GFF) and optimized binding and wash buffers (also provided).

In parallel, total RNA was isolated from the same number of cells with the mirVana miRNA Isolation Kit and with the PARIS Kit (the latter is opimized for longer RNA isolation and does not recover small RNAs efficiently). Comparison of the purified material on a denaturing polyacrylamide gel showed equivalent recovery of small RNA with the mirVana and mirVana PARIS Kits, but RNA species smaller than ~200 nt were absent in the sample processed with the PARIS Kit (Figure 2A). Previous studies [1] demonstrated a correlation between loss of 5S rRNA and tRNA, and inefficient recovery of miRNA or siRNA (see also Figure 3 and 6) using standard GFF protocols. The integrity of the isolated total RNA was assessed on an RNA 6000 Nano LabChip with an Agilent 2100 bioanalyzer. As seen in Figure 2B, RNA isolated with the mirVana PARIS Kit was of high quality (28S/18S rRNA ratio 1.77). The presence of small RNA was evidenced by a clear peak at ~26 seconds.

Figure 2. Analysis of Total RNA Isolated with the mirVana PARIS Kit. Total RNA was isolated from 1 x 106 HeLa cells with the indicated kit as per protocol. (A) 1 g of total RNA was resolved on a 15% denaturing acrylamide gel and stained with ethidium bromide. (B) Electropherogram of total RNA purified
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