MicroRNA Analysis—Start Here (TechNotes 15:3)

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TechNotes 15(3)

MicroRNA Analysis—Start Here

The resources you need for microRNA (miRNA) studies, from isolation through discovery, profiling, quantitation, and validation are available from a single source. Applied Biosystems provides state-of-the-art products to streamline miRNA research, enabling scientists to address the fundamental questions about miRNA function.

The Importance of miRNAs
First referred to as the “biological equivalent of dark matter” [1], miRNAs are small, highly conserved RNA molecules that act as key regulators of development, cell proliferation, differentiation, and the cell cycle. Emerging evidence also implicates miRNAs in the pathogenesis of human diseases such as cancers, metabolic diseases, neurological disorders, infectious diseases, and other illnesses [2]. The active, mature miRNAs are typically 17–24 nucleotide, single-stranded RNA molecules expressed in eukaryotic cells. Recent whole-genome sequencing data indicates that the 5'- and 3'-ends of miRNAs are variable. These alternative length miRNAs are called isomiRs, and their biological function is unknown. miRNAs are known to affect the translation and/or stability of target messenger RNAs. Each miRNA is believed to regulate multiple genes, and it is currently thought that greater than one third of all human genes may be regulated by miRNA molecules [3].

miRNA Fundamental Research Questions
Researchers have only just begun to understand how miRNAs work and what roles they play in biology. Thus, many of the fundamental questions about miRNA have yet to be answered (Figure 1). Many laboratories are now seeking to determine the effects of miRNAs in various experimental systems. Initial inquiries typically strive to characterize which miRNAs are present in a cell type of interest and the differences in miRNA expression among different tissues, developmental stages, or disease states. Additionally, many efforts to discover previously unreported miRNAs are underway. Once miRNAs of interest are identified, subsequent studies may be designed to artificially perturb miRNA expression levels in cultured cells while measuring resulting mRNA, protein, or phenotypic changes. Ultimately, the goal of many projects is to identify the effects of miRNAs on cellular processes, and, as a result, assess their biological impact and significance.

Figure 1. miRNA Fundamental Research Questions.

Powerful Tools for miRNA Research

1. Genome-wide Small RNA Discovery and Profiling
The discovery of new small RNAs is ongoing; 2000 new sequences were added to the miRBase Registry in the last two years [4]. Rapid discovery of unknown small RNAs, including miRNAs and sensitive quantitative small RNA expression analysis, is now possible with Applied Biosystems next generation SOLiD™ System for sequencing. Unlike other methods of detection, which require prior knowledge of the miRNA sequences and may require large amounts of input RNA, the SOLiD System enables the discovery of novel small RNAs from samples as small as 10–500 ng total RNA.

The SOLiD Small RNA Expression Kit is compatible with the Applied Biosystems SOLiD System for seamless integration into the next generation, high throughput sequencing workflow. It provides a simple means to convert the small RNA in a total RNA sample into a library of double-stranded DNA molecules using a robust single-day procedure (see Figure 1 in A Streamlined Workflow for Whole-Genome Discovery and Profiling of Small RNAs). These small RNA libraries can then be fed into the emulsion PCR step of SOLiD System sample preparation. Sequencing the entire population of small RNAs in a sample provides a direct means to identify most, if not all, small RNA species present in the sample including isomiRs and novel miRNAs. The massive data output capability of the SOLiD System provides an efficient way to perform quantitative expression analysis of small RNAs. (See the Genome-Wide Small RNA Discovery and Profiling Workflow.)

2. MicroRNA Discovery and Profiling Using TaqMan® Technology
To identify differentially expressed miRNAs, many researchers first generate a global miRNA expression profile. Megaplex™ Primer Pools, in conjunction with either TaqMan MicroRNA Assay Sets, or TaqMan MicroRNA Arrays, are ideal for such experiments. They can rapidly generate an expression profile for 667, 518, or 303 miRNAs from human, mouse, or rat, respectively, in a single working day (~5 hr) from as little as 1 ng of total RNA. Taking full advantage of the gold standard sensitivity, specificity, and dynamic range afforded by TaqMan Assay chemistry, Megaplex Primer Pools provide significant benefits over microarrays, which require several days and hundreds of nanograms of input RNA to generate data. (See the MicroRNA Discovery and Profiling Using TaqMan® Technology Workflow.)

3. Targeted MicroRNA Quantitation
Follow-up studies on a specific miRNA are often required to validate previous results, or to further explore the function of specific miRNAs. Individual TaqMan MicroRNA Assays can be used to validate results obtained from both small RNA sequencing and from miRNA expression profiling experiments. Through the use of novel adaptations in assay design, Applied Biosystems brings the benefits of TaqMan Assays and quantitative real-time PCR to miRNA detection and quantitation. TaqMan MicroRNA Assays incorporate a target-specific stem-loop, reverse transcription primer. This innovative design addresses a fundamental problem in miRNA quantitation: the short length of mature miRNAs (~22 nt). The stem-loop structure provides specificity for only the mature miRNA target and forms an RT primer/mature miRNA-chimera that extends the 3' end of the miRNA. The resulting, longer RT amplicon presents a template amenable to standard TaqMan real-time PCR. To ensure accurate results, every TaqMan MicroRNA Assay has been functionally validated under laboratory conditions. Applied Biosystems offers a comprehensive collection of TaqMan MicroRNA Assays covering a broad collection of species, including but not limited to human, mouse, rat, Arabidopsis, C. elegans, and Drosophila. In addition, new assays are continually added on a regular basis for human, mouse, and rat in accordance with Sanger miRBase Registry updates. (See the Targeted MicroRNA Quantitation Workflow.)

4. MicroRNA Functional Analysis
Analyses of miRNA function are performed with protocols that are similar to those used for protein-encoding genes. Artificially up-regulating miRNAs can help identify gain-of-function phenotypes; down-regulation or inhibition experiments can be conducted to identify loss-of-function phenotypes. The combination of up- and down-regulation can be used to identify genes and cellular processes that are regulated by specific miRNAs. Further investigation of miRNA function includes studies on miRNA-target interaction and the impact on target mRNA levels and concomitant protein expression. (See the MicroRNA Functional Analysis Workflow.)

TechNotes Archive

RNA Recovery Tailored for miRNA Experiments

Ambion® mirVana™ miRNA Isolation Kit
Samples are lysed in a denaturing lysis solution that both stabilizes RNA and inactivates RNases. The lysate is then Acid-Phenol:Chloroform extracted, yielding a semi-pure RNA sample. The RNA is further purified over a glass-fiber filter to yield either total RNA or a size fraction enriched in miRNAs. This kit uses reagents specifically formulated for miRNA retention to avoid the loss of small RNAs typically seen with standard glass-fiber filter methods.

Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues
The RecoverAll™ Kit procedure requires about 45 minutes of hands-on time and can easily be completed in less than 1.5 hours when isolating RNA. FFPE samples are deparaffinized using a series of xylene and ethanol washes and then are subjected to a rigorous protease digestion with an incubation time tailored for recovery of either RNA or DNA. Nucleic acids are purified using a rapid glass-filter method that includes an on-filter nuclease treatment, and are finally eluted into either water or the low-salt buffer provided. The degree of RNA fragmentation that has already occurred in FFPE tissues cannot be reversed. However, the protease digestion conditions of the RecoverAll Kit are designed to release a maximal amount of RNA fragments of all sizes, including miRNA, in a relatively short amount of time. This RNA can be readily analyzed by real-time RT-PCR and generates profiles equivalent to those seen in RNA isolated from fresh or flash-frozen samples.

TaqMan® MicroRNA Cells-to-CT™ Kit
Start with 10–100,000 cultured cells per sample, either in plates or tubes, and be ready for RT-PCR in 10 minutes. Cells are washed in PBS and lysed for 8 minutes at room temperature; DNase treatment can be performed concurrently. Lysis is terminated by adding Stop Solution and incubating for 2 additional minutes at room temperature. Because samples can be processed directly in culture plates (96- or 384-well), sample handling is reduced, and the risk of sample loss or transfer error is minimized. No heating, washing, or centrifugation is required; the TaqMan® MicroRNA Cells-to-CT™ Kit greatly reduces a traditionally time-consuming, labor intensive process to 10 minutes.

References

1. Bartel DP (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116(2): 281–297.

2. Soifer HS, Rossi JJ, Saetrom P (2007) MicroRNAs in disease and potential therapeutic applications. Mol Ther 15(12): 2070–2079.

3. Kim VN, and Nam JW (2006) Genomics of microRNA. Trends Genet 22(3): 165–173.

4. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res 36: D154–D158.