An RNA isolation system for plant tissues rich in secondary metabolites

Deciphering the underlying mechanisms of gene expression, signal transduction, gene regulation and transcriptome analysis requires a whole gamut of techniques such as northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and construction of cDNA libraries. Substantially pure and un-degraded RNA is a fundamental requisite for all these techniques. A large number of protocols have been developed or extensively modified [1–7], and commercial kits are also available for isolation of RNA from plant tissues. Most of these methods, including kits, were found to be unsuitable for isolation of RNA from Litchi chinensis, Pinus taeda, Pseudotsuga menziesii, Picea glauca, Griffonia simplicifolia and Albizia procera[2–4, 6]. Some of these tissues have phenolic compounds, which get oxidized to form quinones. Quinones bind to RNA and hinder RNA isolation and/or downstream applications [8]. Secondary metabolites often co-precipitate with RNA and affect yield, quality [3] and interfere with downstream applications [9]. Specific protocols developed for such tissues are usually time consuming and also tissue specific [2–7].

Our research work involved cloning of relevant genes from medicinal plants, rheum (Rheum australe) and arnebia (Arnebia euchroma), which are rich in secondary metabolites anthraquinones and alkannins/shikonins, respectively. The prevalent methods namely TRIzol^® (Invitrogen, USA), RNeasy^® (Qiagen, Germany) and guanidinium salt based method [5] either failed to isolate RNA or yielded negligible quantity of RNA from these plants (Figure 1). Three methods we tried utilized a guanidinium-based salt as one of the constituents of the RNA isolation system [1, 5, 10]. A guanidinium-based salt is a strong protein denaturant and inhibitor of RNase. Therefore, it is an ingredient of choice in most of the RNA isolation systems. However several tissues, including those mentioned above were recalcitrant to RNA isolation, possibly due to the presence of guanidinium salts [3, 6, 7]. The presence of secondary metabolites has been attributed to interfere with resuspension of RNA when extracted with guanidinium salts [3]. Since guanidinium salts are also ineffective in dissociating RNA from non-protein complexes, RNA may be lost along with the complex during isolation procedure [11, 12]. It is also likely that the presence of guanidinium salt might promote such a complex formation that would further inhibit RNA isolation. Therefore, it was necessary to develop a composition which is free of guanidinium-based salt and at the same time the composition should possess protein-denaturant activity strong enough to inhibit RNase action.

Compositions free of guanidinium salt have been developed for isolation of RNA [3, 6, 7]. However, these procedures are time consuming, cumbersome, expensive and hence limit simultaneous processing of large number of samples. The present manuscript describes a protocol (IHBT protocol) for isolation of RNA from rheum and arnebia, which does not utilize guanidinium salt and also is simple and rapid. The developed protocol was extended to nineteen more plant tissues with success and the RNA isolated was amenable to downstream applications.

A total of 21 plant tissues were collected either from (i) the wild, (ii) cultivation in the experimental farm of the institute, or (iii) the local market.

A phenol-based, guanidinium salt-free protocol for isolation of RNA was developed. Reagents were selected based on their specific properties: phenol as a strong protein denaturant and inhibitor of RNase; SDS and EDTA are also inhibitors of RNase [18]. This composition provided a cocktail of RNase inhibitors and protein denaturant. Further, pH of the solution was maintained in the acidic range to allow efficient and preferable partitioning of RNA in the aqueous phase leaving DNA in the phenolic phase; DNA prefers basic pH for its partitioning into the aqueous phase [19]. An appropriate concentration of NaOAc was included in the solution to aid precipitation of RNA in the presence of isopropanol. The addition of DEPC-treated autoclaved water after addition of solution I rendered sufficient aqueous environment for partitioning of RNA into the aqueous phase. Our results suggested that the composition developed in the present communication had requisite protein denaturation and RNase inhibitory activity. It appears that the composition and the protocol provided an environment which prevented oxidation of phenolics leading to quick removal of these compounds from the extraction medium. This possibly allowed RNA to be free from quinone/protein complexes, and hence eased extraction and further re-suspension in water.

The developed method successfully isolated RNA from leaf tissues of rheum and arnebia with a yield of 168.66 ± 4.7 and 153.33 ± 11 μg RNA/100 mg of tissue, respectively. This was remarkable in the light that other protocols either failed to yield RNA or yielded extremely low quantity (Figure 1). Though contamination of DNA can not be completely avoided, as is also reported with most of the rapid protocols [20, 21], digestion of RNA with RNase-free DNase took care of the contamination (Figure 2). In fact, DNase digestion is a common step in protocols involving RNA analysis [13–15].

The method so developed was further extended to nineteen more tissues (Figure 3) from different plants having varying amount of secondary metabolites. The A_260/280 ratio of the isolated RNA ranged between 1.8 - 2.0, which indicated the RNA to be relatively free of proteins and contaminants. At times, low A_260/280 ratio ranging between 1.4 - 1.7 was also obtained, particularly when higher amount of the tissue was used. In all the cases, distinct 28 S and 18 S rRNA bands were observed on 1% formaldehyde-agarose denaturing gel indicating intact RNA. Also, the isolated RNA was amenable to downstream applications e.g. RT-PCR, DD, northern analysis, SSH and RACE, as reported in our publications that utilized the present protocol [13, 15–17, 22, 23].

Unlike specific protocols for different tissues, the developed method was very useful in isolation of RNA not only from rheum and arnebia but also from various other plant species belonging to diverse genera. The distinguishing feature of the protocol was the success with those tissues wherein the commonly used protocols failed. The protocol is simple, does not require any specialized material, chemical, instrument and procedure such as ultracentrifugation step through cesium chloride gradient or lithium chloride precipitation, thereby greatly reducing the complications and the time required.