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
Isolation of miRNA begins when total RNA that includes the small RNA fraction is isolated from the samples of interest. However, not all isolation methods retain the small RNA fraction, resulting in loss of miRNAs. Therefore it is important to use RNA isolation methods specifically adapted to retain it. Ambion provides a complete line of RNA isolation kits that allow purification of the total RNA fraction, including small RNAs, from fresh and frozen tissue, from blood, and from formaldehyde- or paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues. Use any of these sources of miRNA containing total RNA for qRT-PCR or Northern analysis. For miRNA profiling by microarray, we recommend that the mature miRNA fraction be purified from any of these total RNA samples and then labeled for microarray analysis. The flashPAGE™ Fractionator, described below, can fractionate mature miRNAs from other small RNA species in just 12 minutes.

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
The mirVana miRNA Isolation Kit, which employs organic extraction followed by purification on a glass fiber filter (GFF), uses specialized binding and wash buffers that are optimized to prevent loss of the small RNA fraction. Unlike other total RNA isolation kits that do not efficiently recover small RNA species <200 nt, the mirVana miRNA Isolation Kit quickly and efficiently isolates total RNA ranging in size from kilobases down to 10-mers (Figure 2). This fraction can then be further enriched for small RNAs <200 nt.

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.

Figure 3. Using the mirVana™ PARIS™ Kit to Analyze RNAi Effects. 1.5 x 106 HUVEC cells were electroporated (800 V, 120 µs, 2 pulses, 0.5 s between pulses) with siRNA (10 µg) targeting GAPDH mRNA or a negative control sequence in 400 µl of siPORT™ siRNA Electroporation Buffer. Total RNA and protein were isolated with the mirVana PARIS Kit 48 hr after electroporation. Total RNA (1 µg) was used to detect the indicated RNA species by Northern blot or with the mirVana miRNA Detection Kit. 32P-labeled RNA probes were prepared by in vitro transcription (mRNA probes, MAXIscript® Kit) or by 5' end labeling (rRNA, miRNA, and siRNA probes, mirVana Probe & Marker Kit). Western blots were performed with 15 µg of total protein and antibodies specific for GAPDH or Ku p70 proteins.

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
The flashPAGE Fractionator System (Figure 4) is a fast, easy, and reliable method for isolating small nucleic acids shorter than 40 nt from total RNA for subsequent miRNA array analysis or cloning applications. This system has been designed and optimized to separate mature miRNA
(19–23 nt) away from longer precursor molecules (Figure 5) and provides an efficient alternative to standard, time-consuming polyacrylamide gel electrophoresis (PAGE) and subsequent gel elution.

Figure 4. flashPAGE™ Fractionator.

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.

Figure 5. Mature miRNA Recovery Using flashPAGE™ Fractionator. To demonstrate recovery of miRNA-sized molecules, Decade™ Markers mixed with 10 µg mouse brain total RNA were loaded onto a pre-cast flashPAGE Gel Cartridge and electrophoresed with the flashPAGE Fractionator. Two successive fractions were collected. For the first fraction, the lower buffer was collected when the indicator dye band had reached the lower end of the gel cartridge (middle lane). After adding fresh lower buffer to the apparatus, the sample was electrophoresed for an additional 10 minutes and the second lower buffer fraction was then collected (right lane). Both buffer fractions were precipitated and loaded onto a polyacrylamide gel for analysis along with a sample of unfractionated Decade Markers (left lane).

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?
Ambion has a large family of miRNA analysis products for sensitive detection of miRNA by a variety of methods. These include primer sets and RT-PCR reagents for the amplification of specific miRNA species (mirVana™ qRT-PCR Primer Sets and the mirVana™ qRT-PCR miRNA Detection Kit), and two distinct miRNA microarray profiling systems: mirVana™ miRNA Probe Set and mirVana miRNA™ Bioarrays. Kits for designing miRNA probes and detecting miRNAs by solution hybridization are also available. For more information on miRNA analysis and profiling, visit the Ambion miRNA Resource Page at www.ambion.com/miRNA.

Figure 6. Selection Guide for Small RNA Isolation and Enrichment.

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