Patricia Powers & Emmanuel Labourier, Ambion, Inc

Quantitative recovery of small RNA such as small interfering RNA (siRNA) and microRNA (miRNA) requires optimized RNA isolation procedures. The mirVana™ miRNA Isolation Kit is the first commercially available kit specifically designed for efficient recovery of small RNA. Developed for cultured cell lines and mammalian tissue samples, the kit can be used to isolate either total RNA or a fraction enriched in RNA species smaller than 200 nt [1]. The mirVana miRNA Isolation Kit is shown here to be a useful tool for small RNA isolation from plant tissues. Procedures for sample collection, storage, and processing to recover high quality RNA are also discussed.

Plant tissues present some unique challenges for RNA isolation, compared to animal tissues and cells. For example, their cell walls may be very difficult to disrupt, and plant cells often contain significant amounts of compounds such as tannins, phenolics, and complex polysaccharides that can affect RNA quality and inhibit downstream reactions. Ambion’s mirVana miRNA Isolation Kit includes features that address many of the issues associated with RNA isolation from plant tissue and allow recovery of high quality and high yields of plant RNA.

Recover Intact RNA from Frozen Plant Tissue

The first step in many plant RNA isolation protocols is to freeze the tissue in liquid nitrogen, and then grind it to a fine powder with a mortar and pestle while frozen. The mirVana miRNA Isolation Kit can be used with both fresh and frozen tissue samples and is, therefore, compatible with most plant RNA isolation protocols. However, freezing and grinding in liquid nitrogen is laborious and time consuming. Furthermore, any accidental thawing will cause the release of endogenous ribonucleases and subsequent RNA degradation.

To simplify the processing of frozen plant tissues, we recommend treatment with RNAlater-ICE which preserves RNA integrity in frozen samples and makes them easier to handle. Animal or plant tissues frozen in liquid nitrogen are simply submerged in cold RNAlater-ICE and allowed to thaw at –20°C (at this temperature, the RNAlater-ICE will be liquid). Thus, tissues thawed in RNAlater-ICE can be dissected, partitioned, and weighed at room temperature; stored at 4°C for several weeks or directly homogenized with a motorized homogenizer (e.g. Polytron) in the Lysis Solution provided with the mirVana miRNA Isolation Kit.

Figures 1 and 2 show high yields of intact RNA containing the small RNA species isolated from several plant tissues that were collected, immediately frozen in liquid nitrogen, and then treated with RNAlater-ICE. Frozen spinach leaves, stored in RNAlater-ICE for more than 18 months, have been found to yield intact RNA, indicating the impressive level of preservation afforded by RNAlater-ICE (Figure 1, right panel).


Figure 1. Total RNA from 4 Plant Tissues Isolated with the mirVana™ miRNA Isolation Kit. RNA was purified with the standard mirVana miRNA Isolation Kit protocol from frozen or RNAlater-ICE treated Arabidopsis thaliana tissues (kindly provided by Vaughan Symonds, University of Texas at Austin, and Dr. Fred Lehle, Lehle Seed Company, Round Rock, TX). Alfalfa, corn, and spinach RNAs were isolated from RNAlater-ICE treated tissues. The spinach tissue was stored for more than 18 months in RNAlater-ICE. 1 µg of RNA was loaded per lane on a 1% denaturing agarose gel.

Recover Pure RNA

Minimize organic compound—RNA cross-linking
Organic compounds such as phenolics, terpenes, and tannins are often found in plant tissues. In living cells these compounds are compartmentalized, but tissue disruption for RNA isolation releases them. Under oxidizing conditions, organic compounds can cross-link with RNA, potentially interfering with quantitation and downstream applications such as reverse transcription and PCR.

The mirVana Lysis Solution is designed to minimize oxidation and to limit free radical-dependent cross-linking to RNA. The purity of plant RNA can be determined by the ratio of spectrophotometer readings at 230, 260, and 280 nm. A ratio of 1:2:1 corresponds with high purity RNA [2]. Arabidopsis thaliana RNA isolated with the mirVana miRNA Isolation Kit typically has an absorbance ratio of 1:1.5:1 and can be used directly in RT-PCR experiments.

Remove polysaccharides
Many plant tissues (e.g. fruits, seeds, tubers) also contain large amounts of polysaccharides that can copurify with RNA. Lithium chloride precipitation can effectively remove polysaccharides; however, it does not efficiently precipitate small RNAs, including miRNA.

The mirVana miRNA Isolation Kit is compatible with Ambion’s Plant RNA Isolation Aid, a specially formulated polyvinylpyrrolidone (PVP) reagent which selectively removes polysaccharides and polyphenolics from tissue lysates during RNA isolation. The reagent is simply mixed with the mirVana Lysis Solution prior to sample homogenization. With A. thaliana samples, including the Plant RNA Isolation Aid in the mirVana Lysis Solution typically results in an improved absorbance ratio of 0.67:2:1.

Enrich for Small RNA

Small RNA analysis often requires extremely large mass amounts of input RNA. Furthermore, techniques for analysis of small RNAs are not compatible with those appropriate for mRNA analysis. For example, small RNAs cannot be analyzed by RT-PCR or standard microarray techniques, but instead are analyzed typically by Northern blot using 15% acrylamide gels or specialized solution hybridization techniques. With the mirVana miRNA Isolation Kit, fractions enriched in RNA species smaller than 200 nt and fractions comprised of the longer RNA species can be prepared from the same sample by using differential binding conditions on the silica filters provided with the kit. This facilitates downstream analysis of both small RNA and mRNA.

A representative small RNA enrichment/depletion experiment is presented in Figure 3. The small RNA-depleted and -enriched fractions were purified from A. thaliana and compared with total RNA isolated from the same amount of tissue. Analyses on denaturing agarose and acrylamide gels demonstrate that small RNAs were quantitatively recovered in the fraction “enriched” for small RNAs. In contrast, longer RNA species such as the actin-2 mRNA were detected only in the fraction “depleted” of small RNAs. Further analysis by Northern blot showed that the same amount of microRNAs miR-163 and miR-167 were recovered in the small RNA-enriched fraction and in the total RNA sample. Since only about 200 ng of the small RNA fraction was loaded on the gel, this corresponded to a 10-fold enrichment relative to the amount of total RNA analyzed (2 µg).


Figure 3. Small RNA Enrichment from A.thaliana. RNA was isolated from the same amount of tissue using either the total RNA or the small RNA enrichment/depletion protocol as described in the mirVana™ miRNA Isolation Kit manual. The same volume of purified RNA was analyzed on a 1% denaturing agarose gel (left panel, equivalent to 1 µg of total RNA) or a 15% denaturing polyacrylamide gel (right panel, equivalent to 2 µg of total RNA). After transfer to BrightStar nylon membrane (Ambion), blots were probed with high specific activity probes prepared either by in vitro transcription with the MAXIscript Kit (actin-2 mRNA) or by 5’ end labeling with the mirVana™ Probe & Marker Kit (miR-163, miR-167).

A Complete Solution

The mirVana miRNA Isolation Kit is part of Ambion’s growing family of tools dedicated to small RNA analysis. Ambion’s mirVana System includes products for isolation of small RNA, preparation of high specific activity probes or small RNA markers, and sensitive detection of miRNA or siRNA.