Isolating miRNA for Profiling Studies
|The first critical step in the analysis of microRNA (miRNA) is purification from a biological sample. Most RNA isolation procedures were developed and optimized to recover total RNA or mRNA while ignoring small molecules. Thus, many of these protocols result in loss of substantial amounts of miRNA and other small RNA species (Figure 1). Ambion has developed a complete line of kits and reagents for isolating miRNAs from a wide range of cell and tissue types, as well as from blood and FFPE samples. These kits enable downstream analyses of miRNAs by array analysis, RT-PCR, solution hybridization, and Northern blotting.|
How to Capture miRNAs
Figure 1. Detection of microRNAs. Not all isolation methods retain the small RNA fraction, resulting in loss of miRNAs. Likewise, not all commercially available total RNAs contain small RNAs. In the report by Grad et al. [Grad et al. (2003) Mol Cell11:1253–1263.], total RNA from a commercially available source was used to detect the presence of miR-200b in various human tissues (by Northern blotting). In this study, miR-200b expression was only detected in human lung tissue (summary of data shown in Panel A). However, it was possible to detect miR-200b in several human tissues using both Northern blot and solution hybridization assay when the total RNA (Ambion’s FirstChoice® Total RNA) was prepared using a method that ensured small RNA retention (B). Interestingly, the levels of miR-200b detected in the lung (the tissue in which miR-200b was previously detected) were lower than that of several other tissues (colon, kidney, pancreas, prostate, and thymus).
Initial Isolation of Total RNA Containing miRNAs: mirVana™ miRNA Isolation Kit and mirVana™ PARIS™ Kit
Figure 2. mirVana™ miRNA Isolation Kit for Efficient Recovery of miRNA. (A) Total RNA was isolated from the same mouse liver lysate using a double phenol/guanidinium extraction (DPGE) or the mirVana miRNA Isolation Kit procedure in triplicate (miRNA 1 to 3). The experiment was performed with two different mouse liver lysates. Each sample RNA (1 µg) was analyzed on a denaturing 15% polyacrylamide gel stained with ethidium bromide. (B) RNAs from the same gel were transferred to a membrane and probed for U2 snRNA and let-7 miRNA. The relative amount of small RNA in each lane was quantified with a phosphorimager. The graph shows the percentage of recovery with respect to the DPGE prep.
The mirVana PARIS Kit permits quantitative recovery of both native protein and all RNA species, including small RNAs, from the same sample (Figure 3). Samples are first homogenized with a special Cell Disruption Buffer that includes nonionic detergent to extract intact protein; a portion of the lysate can be used directly for common applications such as Western blotting, two-dimensional gel electrophoresis, or enzymatic assays. RNA is isolated from the remainder of the lysate using a procedure similar to the mirVana miRNA Isolation Kit.
The small RNA containing total RNA fraction from either the mirVana miRNA Isolation Kit or the mirVana PARIS Kit (or other sources) can be further enriched using the protocol and reagents included in both kits. The total RNA or enriched miRNA is ideal for qRT-PCR and other miRNA detection techniques.
Ambion also has several isolation products for obtaining total RNA that includes the miRNA fraction from special samples. These include kits for isolating RNA from extremely small samples such as LCM tissue, from the leukocyte fraction of whole blood, and from FFPE treated samples. See sidebar, Isolating miRNAs from Special Samples.
Purification of the Mature, Active miRNA Fraction: flashPAGE™ Fractionator System
1 to 100 µg total RNA is loaded onto a flashPAGE Pre-Cast Gel inserted into the flashPAGE Fractionator unit and then electrophoresed through the gel matrix for ~12 minutes. Fractionated samples are then concentrated using a 45 minute protocol found in the flashPAGE Clean-Up Kit or precipitating with ethanol overnight. Extensive validation of the flashPAGE design confirms that greater than 95% of species longer than 40 nt are excluded from the small RNA/DNA fraction when the run is terminated with the included dye. Importantly, faithful representation of small RNAs is maintained.
The flashPAGE Fractionator System includes: (1) the flashPAGE Fractionator unit, (2) pre-cast gels, (3) loading and running buffers, and (4) an optional flashPAGE Clean-up Kit for rapid concentration of fractionated samples.
A guide for selecting miRNA isolation and enrichment products is provided below in Figure 6.
Now that I have my miRNA?
Figure 6. Selection Guide for Small RNA Isolation and Enrichment.