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]. 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.

The role of plant miRNAs

Plant miRNAs have important roles as regulators of plant growth and development. In fact, the majority of known plant miRNAs appear to downregulate transcription factor gene targets, which position miRNAs in the key role of establishing organ patterns and fertility in developing plants (Table 1 compares plant and animal miRNA characteristics). 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. 

Challenges of isolating RNA 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. The Plant RNA Isolation Aid is recommended for use with glass fiber filter-based methods for purification of total RNA from plant tissues, because there are no alcohol precipitation steps, which can co-precipitate nucleic acids with common contaminants found in plant tissue.

The procedure begins with disrupting plant tissue in a guanidinium-based lysis solution (supplied in the mirVana™ miRNA Isolation Kit) to which the Plant RNA Isolation Aid has been added. A brief centrifugation then removes polyphenolic and polysaccharide contaminants before RNA is purified using the glass fiber filter (supplied in the kit). This results in the isolation of total RNA including small RNAs, such as microRNA (miRNA), small interfering RNA (siRNA), and small nuclear RNA (snRNA) from tissues and cells. The kit also provides reagents and a procedure to enrich small RNAs that are 200 bases and smaller and enhance the sensitivity of small RNA detection by solution hybridization, northern analysis, microarrays, and other methods. 

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).



Figure 1. High-quality Arabidopsis thaliana RNA isolated using mirVana™ miRNA Isolation Kit and Ambion® Plant 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. No. 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.




Assays to detect and quantify plant miRNAs

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 detection 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. Our pre-designed TaqMan® MicroRNA Assays can distinguish between closely related miRNAs with as little as one nucleotide difference. Using a stem-looped, gene-specific reverse transcription primer, the assays quantitate only mature miRNAs. The TaqMan® MicroRNA Assays can be used to detect as few as 10 copies of a single miRNA with up to a 7-log dynamic range. Accurate, quantitative results are generated within three hours after obtaining purified RNA.

In this 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).

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.


Choosing the right miRNA assay controls

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

Table 1. Comparison of miRNA from plants and animals.

Plant miRNA Animal miRNA
Almost perfect base-pairing with target mRNA Usually has non-complementary regions with target mRNA
Tend to target coding region Tend to target 3' untranslated region
Major mechanisms of action: cleavage of mRNA target or inhibition of transcription Major mechanism of action: inhibition of translation
Often belong to large miRNA gene families
Often conserved among plant species

Reference

1. 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.