Isolate and Quantitate miRNA from Plants
MicroRNA (miRNA) quantitation can be used for a myriad of downstream applications, including validation of predicted miRNAs, tissue expression profiling, comparison with mRNA expression, and biomarker discovery. However, isolating and quantifying miRNA from plant samples can be technically difficult. This article describes successful results obtained with Arabidopsis thaliana miRNA isolated using the Ambion® Plant RNA Isolation Aid in conjunction with the mirVana™ miRNA Isolation Kit. The resulting miRNA was quantitatively analyzed using TaqMan® MicroRNA Assays [1].
Although the role of miRNA-regulated gene expression in plant biology is just beginning to be appreciated, many ongoing studies are directed toward understanding the scope and mechanism of miRNA-mediated effects in plants (see sidebar, Brief Overview of Plant miRNAs). With the availability of straightforward methods for extraction of plant total RNA (including the small RNA fraction) and sensitive real-time RT-PCR assays for miRNA, the stage is set for rapid progress that promises to yield important insights in botanical science.
RNA Isolation from Plants
One of the major issues with plant RNA isolation is the presence of problematic biomolecules, such as polyphenolic compounds and polysaccharides. The Ambion Plant RNA Isolation Aid eliminates many of these contaminants with a pre-extraction spin step and the use of polyvinylpyrrolidone, a high molecular weight polymer that binds to polyphenolics and polysaccharides (see sidebar, Reagents and Kits for Studying Plant miRNAs).
As shown in Figure 1, high-quality Arabidopsis thaliana RNA can be obtained using the Plant RNA Isolation Aid in conjunction with the mirVana miRNA Isolation Kit. In addition to the 18S and 28S ribosomal RNA (rRNA) bands, the chloroplast and mitochondrial rRNA bands common to plant samples are also present (Figure 1).
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| Figure 1. High Quality Arabidopsis thaliana RNA Isolated Using mirVana™ miRNA Isolation Kit and Plant Ambion® RNA Isolation Aid. RNA was isolated using the mirVana miRNA Isolation Kit and the Ambion Plant RNA Isolation Aid. (A) RNA (150 ng) was assessed by capillary electrophoresis (Agilent® 2100 bioanalyzer). (B) RNA (1 µg) was also assessed on a 1% denaturing agarose gel (NorthernMax® Kit, Cat# AM1940). Note the bands represent 18S and 28S rRNA (two largest bands), as well as chloroplast and mitochondrial rRNA. Kahl G and Meksem K, eds. (2008) The Handbook of Plant Functional Genomics, “Real-Time Quantitation of MicroRNAs by TaqMan® MicroRNA Assays,” Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. Reproduced with permission. |
MicroRNAs in Plants
Short sequences and high homology within large miRNA gene families make plant miRNAs difficult and unreliable targets for traditional hybridization-based detection methods. The development of mature miRNA-specific qRT-PCR has enabled easier miRNA dection and opened the door for better detection of very low-abundance miRNAs, some of which may be expressed only during narrow developmental timepoints.
TaqMan® MicroRNA Assays, used with the TaqMan MicroRNA Reverse Transcription Kit, enable quantitation of miRNAs with specificity and unsurpassed sensitivity (see sidebar, Reagents and Kits for Studying Plant miRNAs). For example, fifty TaqMan MicroRNA Assays were run with A. thaliana total RNA, and the data clearly demonstrate a range of expression levels (Figure 2). Moreover, since many miRNAs are conserved among different plant species, assays designed for such targets can be readily applicable for use across multiple species. For example, A. thaliana miRNA assays have been used for miRNA detection in maize (not shown).
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| Figure 2. TaqMan® MicroRNA Assays Using RNA from Arabidopsis thaliana Seedlings. Fifty TaqMan MicroRNA Assays were used to assess RNA from A. thaliana seedlings. Total RNA was isolated as described in Figure 1. This experiment demonstrates the range of expression levels seen for the miRNAs. The results from three input quantities of total RNA (0.07–7 ng) show the sensitivity and reproducibility of the assays. Kahl G and Meksem K, eds. (2008) The Handbook of Plant Functional Genomics, “Real-Time Quantitation of MicroRNAs by TaqMan® MicroRNA Assays,” Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. Reproduced with permission. |
Control Assays
When considering endogenous controls suitable for normalizing data from TaqMan MicroRNA Assays, it is important that they share similar properties to miRNAs, such as RNA size and compatibility with miRNA assay design. After analyzing 32 A. thaliana non-coding RNAs, stable expression patterns were observed for snoR41Y, snoR65, snoR66, and snoR85. In addition to snoRNA controls, we recommend using: 1) endogenous miRNA genes; 2) structural RNAs (e.g., 18S rRNA, U6 snRNA); and 3) housekeeping genes (e.g., GAPDH, actin, β-tubulin). Several control genes should be tested, so that at least two can be chosen to normalize your experiment.
To see a detailed protocol, or for more information about plant genomics, refer to The Handbook of Plant Functional Genomics [1].
Scientific Contributors
Toni L Ceccardi and Caifu Chen • Applied Biosystems, Foster City, CA
Marianna M Goldrick and Rick C Conrad • Applied Biosystems, Austin, TX
Peifeng Ren • BASF Plant Sciences L.L.C., Research Triangle Park, NC
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| Figure 3. Comparison of miRNA from Plants and Animals. |
For Research Use Only. Not for use in diagnostic procedures. |
