Isolation and Detection Methods of Plant miRNAs.

Small RNAs (sRNAs) are RNAs of low abundance in organisms. Among sRNAs, miRNAs are included and represent approximately 10% of the total number of sRNAs. The isolation of sRNAs is critical for miRNA detection and analysis. The precipitation of low-molecular-weight (LMW) RNAs from total RNA extracts has allowed enrichment of sRNAs. Here, we describe a simple method to isolate sRNAs from different plant species. The main advantage of this method is that it does not need first an extraction of total RNA and it is not based on TRIzol® reagent. This method has been successfully used for miRNA analyses by Northern blot assay and RT-qPCR (these techniques are as well described in this chapter), as well as sRNA library preparation.

Northern Blot Analysis of microRNAs and Other Small RNAs in Plants.

The study of regulatory small RNAs, such as siRNAs and microRNAs in plants, has necessitated methods tailored to their unique features. Their analysis demands the use of sensitive and quantitative methods for their detection. The use of Northern blot hybridization offers an attractive alternative to address qualitative as well as quantitative features. We highlight the advantages and shortcomings of this method and offer a detailed description of the techniques that best work in our hands, considering their use for the study of several small RNAs in multiple samples. We enumerate relevant details as well as cautionary comments in cases where we have detected potential difficulties.

A dicistronic precursor encoding miR398 and the legume-specific miR2119 coregulates CSD1 and ADH1 mRNAs in response to water deficit.

Plant microRNAs are commonly encoded in transcripts containing a single microRNA precursor. Processing by DICER-LIKE 1 and associated factors results in the production of a small RNA, followed by its incorporation into an AGO-containing protein complex to guide silencing of an mRNA possessing a complementary target sequence. Certain microRNA loci contain more than one precursor stem-loop structure, thus encoding more than one microRNA in the same transcript. Here, we describe a unique case where the evolutionary conserved miR398a is encoded in the same transcript as the legume-specific miR2119. The dicistronic arrangement found in common bean was also observed in other legumes. In Phaseolus vulgaris, mature miR398 and miR2119 are repressed in response to water deficit, and we demonstrate that both are functional as they target the mRNAs for CSD1 and ADH1, respectively. Our results indicate that the repression of miR398 and miR2119 leads to coordinated up-regulation of CSD1 and ADH1 mRNAs in response to water deficit in common bean and possibly in other legumes. Furthermore, we show that miRNA directed CSD1 and ADH1 mRNAs up-regulation also occurs when common bean plants are exposed to flooding, suggesting that plant redox status and fermentation metabolism must be closely coordinated under different adverse conditions.