A simple and rapid method for isolating high-quality RNA from kenaf with high polysaccharide and polyphenol contents

Obtaining RNA of high purity and integrity is important for conducting analytical studies, such as reverse transcription, qRT-PCR, northern blot and complementary DNA (cDNA) library construction, in plant molecular biology. However, isolation of high-quality RNA from higher plant tissues is a challenging process due to the interference of endogenous RNase activation and external RNase introduction. In particular, tissues that are rich in polysaccharides, polyphenolic compounds and other types of secondary metabolites complicate RNA isolation [1]. Polysaccharides are visually evident by their viscous, glue-like texture and make the pipetting of nucleic acids unmanageable [2], and they can also coprecipitate with nucleic acids and constitute the major hurdle for RNA isolation [3]. The coprecipitation of these compounds with RNA reduces yield and increases the possibility of rapid degradation, making the sample unsuitable for further downstream applications due to the severe inhibition of enzymatic activity [4–7]. In turn, polyphenols are known to be readily oxidized to form quinones that can irreversibly interact with proteins and nucleic acids to form high-molecular-weight complexes that hinder isolation of good-quality RNA [8]. Furthermore, with maturity or stressful plant growth conditions, tissues contain increased quantities of polyphenols and polysaccharides, which may further encumber the isolation of high-quality RNA [9]. Hence, with the removal of such components is necessary to achieve the isolation of RNA of high quantity and quality. Many specific protocols, including those utilizing cetyltrimethylammonium bromide (CTAB)/NaCl [10], CTAB/lithium chloride (LiCl) [7], TRIzol [11] and sodium dodecyl sulfate (SDS) [12], designed for plants with high content of polysaccharides and polyphenols, and have been developed and used to extract high-quality total RNA from young leaves [13]. The majority of them, however, have certain limitations, as they are often expensive [14], tissue specific [11], time consuming [15], or technically complex [16].

Kenaf (Hibiscus cannabinus) is an important fiber crop that is widely used in paper-making and weaving [17] and harbors significant heterosis in terms of phloem fiber production. Therefore, understanding the genetic factors underlying heterosis in kenaf holds promise for its breeding and production. Nucleic acid extraction from kenaf is notoriously difficult because kenaf has high concentrations of polysaccharides and polyphenols, which could coprecipitate with RNA and inhibit the enzymatic reactions in subsequent steps [16]. Several protocols, such as those of commercial kits, have been reported for RNA isolation from kenaf tissues [14,18]. However, the obtained RNA was contaminated by polysaccharides and easily degraded by residual RNase that could not be inhibited. Furthermore, although the extraction of nucleic acids was shortened using commercial reagent kits compared to conventional CTAB methods, these are not only expensive, but also contaminated by phenolic compounds, saccharides and proteins, resulting in low-quality RNA that is unsuitable for downstream applications.

Here, we present a simplified, efficient and cost-effective method for the isolation of high-quality total RNA from kenaf anthers, petals, leaves, stems and roots. For comparative purposes, we established a protocol for RNA extraction using CTAB in the extraction buffer, LiCl for precipitation [19], and a commercially available ready-to-use reagent (Huayueyang, Beijing) for nucleic acid extraction. In contrast to the other tested methods, the RNA prepared by the present protocol demonstrates a significant improvement in time savings and economics; In addition, the RNA was of high quality, extracted in high quantities and successfully used for downstream applications such as RT-PCR, qRT-PCR and northern blot analysis. Subsequently, this proposed protocol was successfully used for RNA extraction from other plants, such as cotton, roselle and soybean.

Materials & methods

Plant material

Kenaf was cultivated in the test field of Guangxi Academy of Agricultural Science (Nanning, Guangxi, China), and leaves, stems, roots, petals and anthers were collected at the flowering stage. Both cotton anthers and roselle calyxes were collected at the flowering stage as well. Soybean stem were harvested from 60-day-old plants grown in the field. All samples were flash-frozen in liquid nitrogen and stored at -80°C for further use.

Nucleic acid extraction

Preparation

Prior to RNA extraction, mortars and pestles were wrapped in tin foil and baked in an oven at 180°C for 6 h. All glassware was treated with 0.01% (v/v) diethylpyrocarbonate (DEPC) and autoclaved, and consumables (tips and tubes) were certified as RNase-free. All chemicals used were of molecular biology grade and purchased from Solarbio (Beijing, China). To limit exposure to noxious components (e.g., β-mercaptoethanol [B-ME], guanidine thiocyanate and phenol-chloroform), RNA extraction was conducted in a fume hood.

Solutions & reagents

The RNA extraction buffer was composed of 100 mM Tris (pH 8.0), 25 mM EDTA (pH 8.0), 2 M NaCl, 2% CTAB (w/v) and 7% B-ME (V/V)¹. Other solutions used in this study included a saturated guanidinium isothiocyanate solution (5 M), 100 and 75% (v/v) ethanol, RNase-free distilled deionized water, and water-saturated phenol:chloroform:isoamyl alcohol (PCI) 25:24:1 (v/v/v). All solutions were prepared with 0.01% DEPC-treated distilled water. RNA isolation was carried out twice using independent pools of tissue samples.

Nucleic acid isolation procedure

Nucleic acid isolation was carried out using the following steps:

The other two comparative RNA extraction methods included the protocol described by Zhou et al. [19] and a ready-to-use RNA extraction kit (Huayueyang, Beijing, cat no. 0416-50) that was implemented following the manufacturer's instructions. Each kenaf tissue type (anthers, petals, mature leaves, roots and stems) was extracted in two independent experiments and measured by each method.

Assessment of nucleic acid quantity & quality⁴

The purity and concentration of our RNA extracts were determined using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, MA, USA) by measuring absorbance ratios of A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀. The integrity of total RNA was verified by running a 500-ng sample in a 1% (w/v) agarose gel that was then stained with SYBR Green II (Tiandz, China) and imaged using a UV transilluminator from Syngene (Bio-Rad, NJ, USA).

DNase & RNase treatment

The gDNA remover that contain in the first strand cDNA synthesis kit (TransGene Biotech, Beijing, China) was used to eliminate the DNA that contaminated in the RNA samples according to the manufacturer's protocol. In turn, DNA from genomic samples was treated with RNase A (Solarbio, Beijing, China) to remove residual RNA.

RT-PCR, real-time RT-PCR & northern blots

The isolated RNA samples were reverse transcribed to confirm downstream amenability. In total, 1 µg of total RNA was used in a reverse transcription reaction using TransScript One-Step gDNA removal and cDNA synthesis SuperMix (TransGene Biotech, Beijing, China) to obtain 20 μl of cDNA solution following the method published by [20]. The cDNA was PCR amplified by cox2, in which primers were designed to span introns and amplify a 717-bp fragment for cDNA and a 2208-bp fragment for gDNA, including a 1491-bp intron (primers are shown in Table 1). PCRs were carried out in a final volume of 20 μl of reaction mixture with 2 × Taq Master Mix (Vazyme, China) using 50 ng of reverse transcriptase product as a template, and 0.3 μM of each forward and reverse primer. The thermal cycling program for PCR amplification was used as described by Liao et al. [21]: predenaturation at 94°C for 3 min, followed by 35 cycles of 40 s at 94°C for denaturation, 1 min at 58°C for annealing, 2 min at 72°C for extension, and a final step of 5 min at 72°C. The amplified product was visualized by gel electrophoresis in 1% (w/v) agarose gel. We also presented some samples for the expression of the mitochondrial genes cox3 and atp9 using real-time RT-PCR and northern blot analysis following the procedure described by Liao et al. [22]. All primer sequences used in this study are listed in Table 1.

Note: The annotation for the corresponding superscript numerals is as follows:

Results & discussion

Quantity, quality & yield of total RNA

Several total RNA extraction protocols specifically designed for kenaf have been reported. However, these protocols present important limitations: many reported methods utilize expensive commercial extraction kits [14,18] and others are time consuming, which could cause a potential risk of RNA degradation in the process of extraction [19], while other kenaf-based protocols are for specific tissues [23]. In the current study, we described a rapid, efficient and reliable protocol that allowed for the extraction of high-quality total RNA from several kenaf tissues. With our method, we successfully isolated high-quality RNA from kenaf anthers (Figure 1A: lanes 1–2), sepals (Figure 1A: lanes 3–4), mature leaves (Figure 1A: lanes 5–6), roots (Figure 1A: lanes 7–8) and stems (Figure 1A: lanes 9–10). RNA samples showed two sharp and well-resolved ribosomal bands corresponding to the 28S and 18S rRNAs on 1.0% agarose gels (Figure 1A), and the 28S rRNA band was twice as abundant as the 18S rRNA band (Figure 1A), indicating that little or no RNA samples isolated from the different tissues were degraded during extraction. Spectrophotometric analysis revealed A₂₆₀/_A280 ratios ranging between 2.11 and 2.15 (Table 2), indicating a lack of protein contamination. Similarly, the A₂₆₀/A₂₃₀ ratios of all tested samples were greater than 2.0, indicating that the obtained total RNA was highly pure and free of protein, polyphenol and polysaccharide contamination [7,11,13]. The yields of total RNA extracted from different kenaf tissues were quite diverse. The kenaf anthers yielded the highest amount of total RNA (229.31 μg/g fresh weight [FW]), and the root issues displayed the lowest yield levels (76.24 μg/g FW) (Table 2), indicative of the higher number of RNA cells in kenaf anthers than in roots, resulting in high sample recovery.

Comparison with other RNA extraction methods

Nucleic acids isolated from kenaf tissues following the proposed protocol were compared both in quantity and quality with those isolated from the LiCl-based protocol proposed by Zhou et al. [19] and the ready-to-use RNA extraction kit (Huayueyang, Beijing). The quantity and purity of kenaf RNA preparations when using this simple protocol were found to be superior to those obtained using the previously reported protocols (Table 2 & Figure 1). As shown in Table 2, according to the NanoDrop spectrophotometric quantification, the total nucleic acid quantity was the highest when using the proposed protocol, followed by similar amounts when using the RNA extraction kit, while the yield was the lowest when using the method described by Zhou et al. [19]. However, gel electrophoresis showed that nucleic acids extracted from kenaf tissues were nearly degraded, especially when using the Li-Cl-based protocol described by Zhou et al. (Figure 1B & C) [19]. Nucleic acid purity indicated by A₂₆₀/A₂₈₀ ratios was between 1.48 and 1.94 for the LiCl-based and RNA extraction kit protocols, respectively. This suggested that the high yields shown by spectrophotometric analysis were likely the result of false measurements of secondary metabolite contaminants [24], as polysaccharides and polyphenolic compounds often coprecipitate and contaminate nucleic acids during isolation, thereby affecting both the quality and quantity of isolated nucleic acids [25]. Similarly, A₂₆₀/A₂₃₀ absorbance ratios were lower (ranging between 0.55 and 1.41) following these two protocols, indicative of contamination with polyphenols and polysaccharides [26].

In the proposed method, CTAB, a strong ionic denaturing detergent, at a final concentration of 2% (w/v) and a high concentration of B-ME (7% (v/v)) were added in the extraction buffer. This could completely solubilize the cell membranes and bind to the nucleic acid, as well as eliminate most of the polysaccharides and polyphenolic compounds [26,27]. In addition, a relatively high NaCl content (2 M) in the extraction buffer promoted salting out of the protein and avoided coprecipitation of polysaccharide with RNA while leaving RNA in the solution [28]. Moreover, guanidinium isothiocyanate is an RNase inhibitor that can effectively inhibit the activity of RNase during extraction [29]. Furthermore, the presence of precooled 5 M guanidinium isothiocyanate and absolute ethyl alcohol that could bind to nucleic acids formed a jelly-like precipitate that could then be adsorbed by an RNA purification column. Taken together, these results show that good-quality total RNA was successfully obtained from kenaf tissues with high levels of secondary metabolites. The current protocol of RNA isolation was not only efficient but also required less time (∼1.5 h) than earlier methods described by Chen et al. [23] and Zhou et al. [19], which required approximately 8 h. Simultaneously, lower amounts of reagents were used throughout the current procedure, thus contributing to reduced costs.

Reverse transcription & downstream applications of RNA

The RNA isolated by the proposed method was tested by amplifying the cox2 gene fragment. A 2208-bp fragment was amplified using genomic DNA as a template, and a 717-bp fragment was amplified using cDNA as a template (Figure 2). This suggested that the genomic DNA was successfully eliminated from all samples by the gDNA remover reagent during reverse transcription, and PCR products using cDNA as the template indicated high-quality RNA extraction. Furthermore, this differential amplification of fragments in genomic and cDNA samples not only proved the quality of isolated RNA samples and the absence of DNA contamination, but also confirmed the amenability of this method for downstream processing without any ambiguity. Moreover, the isolation of RNA with the current protocol was successfully employed for real-time quantitative PCR (qRT-PCR) gene expression analysis and northern blot analysis. As shown in Figure 3, the values of qRT-PCR cycle thresholds (Ct) ranged from 17 to 25 cycles (Figure 3A & B) and the melting curve was specific, with a solitary peak occurring at approximately 82–83°C (Figure 3C–F). In addition to qRT-PCR, northern blot analysis was carried out to further demonstrate the quality of RNA prepared by our protocol. The fluorescent signal with clear bands and high intensity was detected in RNA transcripts (Figure 4), indicating that high-quality RNA was isolated from kenaf tissues and was suitable for downstream application.

The present protocol could also be successfully used for RNA extraction from other recalcitrant plant tissues, such as cotton and roselle. The consistency of results obtained from independent biological replicates confirmed the repeatability and reproducibility of this method and provided a useful tool for molecular studies focusing on crops rich in secondary metabolites. This protocol has been also employed successfully using soybean stems as material (Figure 5), therefore having a wide range of applicability. In addition, the proposed protocol is also suitable for genomic DNA isolation following treatment with RNase A after initial nucleic acid extraction. Agarose gel electrophoresis revealed that the intact and pure of genomic DNA was obtained without any contamination of proteins, polysaccharides and polyphenolic compounds (Figure 6). Accordingly, these results indicated that the proposed protocol had great potential to be used for rapid isolation of genomic DNA, which is of sufficient quantity and quality for downstream PCR applications.

Conclusion

We reported a new protocol for the extraction of high quantities of high-quality RNA from kenaf tissues with high levels of polysaccharides and polyphenols, and the extracted RNA was suitable for subsequent gene isolation and expression experiments, such as reverse transcription, qRT-PCR and northern blot analysis. This protocol is a reproducible, simple, inexpensive and time-efficient method to isolate RNA from kenaf, especially from tissues such as anthers, petals and leaves, which are rich in polyphenols and polysaccharides. Thus, this simple and fast protocol has been a routine protocol in our laboratory for RNA isolation from cotton, roselle, soybean stems and other plants with high levels of secondary metabolites. In addition, a high quality and quality of gDNA was obtained from the isolated nucleic acic by treatment with RNase A. This protocol greatly reduces costs and labor time without compromising the quality and yield of RNA samples.

Future perspective

Molecular design breeding based on plant gene function is a crucial direction for the future development of the agricultural and seed industry. The acquisition of high-quality RNA is an indispensable prerequisite for the analysis of plant gene function. A convenient, time-saving and cost-effective method is necessary for scientists to acquire high-quality RNA for their specific targets research of interest. Beyond our method, with the continuous expansion and improvement of genomic databases, gene function annotation based on public databases will be more accurate in the next 5–10 years.