A modified method for efficient RNA isolation from mangrove root tissues rich in secondary metabolites

Background

Mangroves are a polyphyletic group of halophytic woody plants restricted to tropical and subtropical intertidal zones [1]. They are further classified into true mangroves and mangrove associates based on salt-tolerant traits and ecological adaptations [2]. Kandelia candel (L.) Druce and Rhizophora mucronata Lam. are true viviparous mangroves that belong to the Rhizophoraceae family [3,4]. Among the mangroves selected for this study, R. mucronata and K. candel are non-salt-secreting species [5]. Mangroves provide essential goods and ecosystem services such as the prevention of soil erosion, sediment stabilization, nutrient supply and regeneration, flood regulation, timber, food, medicine and habitat [6]. However, mangroves are vulnerable to weather events such as hurricanes, typhoons and cyclones [7]. Although mangrove forests are a valuable asset to the coastal ecosystem, there are only a few studies investigating their gene regulation and functional adaptation to harsh intertidal zones. Molecular-level study of apoplastic barrier formation in Avicennia officinalis revealed the importance of salt exclusion in roots [8,9]. Recent studies have elucidated the genetic mechanism of salt glands in Avicennia marina, a highly salt-tolerant mangrove species, with the help of transcriptomic studies [10,11]. Comparative transcriptome analysis between Acanthus illicifolius and its terrestrial relatives found an evolutionary link leading to adaptation in intertidal environments [12]. Tissue-specific transcriptomic studies in A. marina have found halotolerant genes that can be used to enhance plant stress tolerance [13]. Similarly, transcriptome analysis of mangrove plants has identified genes related to abiotic stress tolerance and epigenetic changes [14–16]. Chen et al. [17] studied the role of small RNA in conferring salinity tolerance to Sonneratia apetala, a mangrove species widely used in conservation activities. Small RNAs regulating homeostasis were identified in Rhizophora apiculata [18]. Similar research on small RNA in Bruguiera gymnorrhiza and K. candel showed that mangroves and glycophytes differ in their miRNA expression profile [19]. The epigenetic profile of mangroves and their role in salinity tolerance is less explored. As there are no studies related to small RNA expression in R. mucronata, elucidating transcriptomic and epigenetic expression could provide information on salinity tolerance and insights into the status of mangrove populations. Detailed studies on the molecular basis of stress tolerance will provide insights into their adaptation mechanisms and helpful information for conservation activities and conventional breeding processes for crop productivity [16,20]. For example, the expression of the CYP94B1 gene from A. officinalis conferred salt tolerance to rice and Arabidopsis seedlings [9]. Salinity tolerance capacity and establishment of seedlings in stressed zones differ among each mangrove species. Thus, studying their tolerance level, growth rate and distribution pattern will help identify vulnerable populations [21]. A study of the transcriptomic responses of Kandelia obovata after chilling stress identified positively selected genes with a role in abiotic stress resistance [15], and this can be used to study the status of rehabilitated mangroves. A better understanding of mangrove adaptive mechanisms to stressful intertidal environments will aid in precise conservation planning.

Issues in RNA extraction from mangrove plants

Transcriptomic data provides gene expression patterns of plants under stress and is helpful in elucidating pathways related to stress tolerance [11,22]. Higher yield and quality of isolated RNA are necessary to proceed with downstream experiments such as cDNA synthesis, real-time quantitative PCR (RT-qPCR), RNA sequencing and microarray analysis. Often, RNA isolation methods cannot extract high-purity RNA due to the coprecipitation of metabolites and degradation by RNase [23,24]. Isolation of RNA from Cupressaceae plants is difficult due to polysaccharides and polyphenol contamination [24]. Similarly, RNA isolation is difficult due to the abundance of polyphenols and polysaccharides in Dendrobium huoshanense [25]. Different extraction methods have been tried to isolate good-quality RNA from mangroves, as they are rich in secondary metabolites [26,27]. The polysaccharides in plant tissues precipitate along with RNA, whereas secondary metabolites bind to proteins and RNA to form complexes, compromising overall quality and yield [28].

Extraction of high-quality RNA from salt-tolerant mangroves becomes challenging owing to the accumulation of reactive oxygen species, specialized metabolites, proteins and enzymatic degradation of RNA. Hence, modifications in routinely used protocols are necessary to obtain intact RNA without the interference of metabolites [28]. For example, proteinase K and β-mercaptoethanol eliminate RNase and protein content [29]. The use of β-mercaptoethanol as a reducing agent prevents the oxidation of phenolic compounds; thus, coprecipitation of oxidized products with RNA during isolation is reduced [26,30,31]. Spermidine is also used to remove RNases [29]. Polyvinylpyrrolidone (PVP) helps in the removal of phenolic compounds [25,27]. Moreover, soluble PVP for RNA isolation is suitable for removing proteins [27]. Lithium chloride (LiCl) helps separate DNA and polysaccharides in the aqueous phase, while interaction with the sugar-phosphate backbone precipitates the RNA [32–37]. Thus, the overall yield can be improved by using chemical substances such as LiCl, β-mercaptoethanol and proteinase K [27,29,33].

RNA extraction methods and kits that have been commonly used in mangroves are based on cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate and TRIzol [23,35,38]. Kit-based methods are routinely used to extract high-quality nucleic acids from plant tissues [16,39]. Several studies have reported that commercial kits were unsuccessful in isolating quality RNA from mangrove tissues [23,26]. The advantage of protocol-based RNA extraction methods is that they can be modified according to the sample requirements. In RNA isolation, SDS and CTAB are used for cell disruption, while β-mercaptoethanol is used to prevent oxidation [23,30,40]. A method using phenol-chloroform separation, LiCl and sodium acetate precipitation successfully isolated RNA from mangroves [23]. Although the RNA quality obtained from mangrove foliage was reported to be superior to the RNA quality obtained from roots in the phenol (HiMedia, Mumbai, India) and chloroform (Sigma-Aldrich, MO, USA) based method [23], the same could not be extracted from root samples of the mangrove plants viz. K. candel and R. mucronata. The method described by Rubio-Piña and Zapata-Pérez [23] yielded comparatively lower concentrations of RNA from the root than the stem and leaf. Moreover, good-quality RNA could not be isolated from mangrove roots with the most commonly used kit, RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and TRIzol™ Reagent (Invitrogen, CA, USA) [23,26]. Thus, the use of phenol-chloroform in isolating RNA from mangrove root tissues was not successful. A slightly different RNA isolation approach by Kiefer et al. [29] was performed with modifications. Modifications include the exclusion of spermidine (Sigma-Aldrich, MO, USA) from the extraction buffer to prevent the formation of white precipitate in the extraction steps. Furthermore, the expensive reagent Nucleon PhytoPure resin was excluded from the method of Kiefer et al. [29] and proteinase K (HiMedia, Mumbai, India) was added to the extraction buffer sample preparation. As mentioned by Kiefer et al. [29], the addition of proteinase K (HiMedia, Mumbai, India) helps to remove protein contamination. Although the yield was higher than other methods in the modified protocol of Kiefer et al. [29], RNA suitable for downstream applications could not be isolated. Primary and secondary plant metabolites in mangrove root tissues might have affected RNA quality. Therefore, an optimized method was developed based on Kiefer et al. [29] with the addition of LiCl (Sigma-Aldrich, MO, USA) and sodium acetate (Sigma-Aldrich, MO, USA) to obtain better RNA yield from mangrove root samples.

Current protocol

The present study used a Nucleospin RNA Plant and Fungi isolation kit (Macherey-Nagel, Düren, Germany) and CTAB extraction buffer-based protocols with modifications to obtain high-quality RNA from the root tissues of mangrove species- K. candel and R. mucronata. CTAB extraction buffer-based protocols were not able to isolate high-quality RNA from K. candel or R. mucronata. The Nucleospin RNA Plant and Fungi isolation kit (Macherey-Nagel, Düren, Germany) uses silica membrane and binding based technology to obtain higher RNA concentrations. But the kit was only able to isolate RNA from K. candel. The current optimized protocol is based on the method of Kiefer et al. [29], which uses chloroform-isoamyl alcohol to extract RNA. The addition of proteinase K (HiMedia, Mumbai, India) as suggested in the original protocol [29] considerably increased the purity of isolated RNA. To avoid contamination with excess phenol, chloroform-isoamyl alcohol extraction was chosen. Fu et al. [26] isolated RNA from leaves of five different mangrove species without phenol-chloroform separation and the average RNA yield was 20–40 μg/g fresh weight (FW), which is less than the previous report on mangroves [23]. RNA yield can vary between species. In this modified protocol, LiCl (Sigma-Aldrich, MO, USA) was used to precipitate RNA, and the step was performed for an extended period to obtain a higher yield. The pellet obtained was subjected to DNase I (New England Biolabs, MA, USA) degradation of contaminating DNA. Sodium acetate (Sigma-Aldrich, MO, USA) and ethanol (HiMedia, Mumbai, India) precipitate RNA by making it less hydrophilic and the remaining components can be removed by ethanol wash [41–43]. In this modified method, the precipitation step was followed by ethanol wash to minimize the loss of RNA, which ensures purity and integrity. In contrast, Rubio-Piña and Zapata-Pérez [23] did phenol-chloroform extraction after precipitation and DNase treatment. With the previously described changes, this modified protocol extracted RNA from both K. candel and R. mucronata with better concentration and purity. The plant RNA isolation kit and modified protocol gave high-quality RNA. The total RNA obtained was visualized using 1.2% denaturing agarose gel electrophoresis. RNA quality was assessed using Agilent Tapestation, followed by cDNA library preparation and transcriptome sequencing. Further, RT-qPCR of three genes was performed with RNA isolated from K. candel and R. mucronata.

Materials & methods

Plant material

Viviparous propagules (Figure 1) of K. candel and R. mucronata were collected during fruiting seasons from the Karingode riverbank near Padannakkad, India (12°15′34.5″N 75°07′09.2″E) and Mogral Puthur, Kasaragod, India (12°33′47.3″N 74°57′29.7″E), respectively. Propagules were planted in pots containing a sand-compost mixture and grown for a month. The root samples were wrapped in aluminum foil, snap-frozen in liquid nitrogen and kept at -80°C. All glassware, plasticware and tips used in the experiments were autoclaved. The mortar and pestle were kept at 250°C for 2 h and were used once cool. Reagents and extraction buffers were prepared with diethylpyrocarbonate (HiMedia, Mumbai, India)treated water.

Reagents & extraction buffers

Extraction buffer for the optimized protocol was prepared as follows: 100 mM Tris-HCl (pH 8.0) (HiMedia, Mumbai, India); 25 mM ethylenediamine tetraacetic acid (EDTA) (HiMedia, Mumbai, India); 2 M NaCl (HiMedia, Mumbai, India); 2% CTAB (w/v) (HiMedia, Mumbai, India); 2% PVP (w/v) (HiMedia, Mumbai, India); and 2% β-mercaptoethanol (v/v) (HiMedia, Mumbai, India) were added to the extraction buffer just before use. Chloroform (Sigma-Aldrich, MO, USA) and isoamyl alcohol (HiMedia, Mumbai, India) at the ratio of 24:1 for separation, LiCl (8 M) (Sigma-Aldrich, MO, USA) and sodium acetate (3 M) (Sigma-Aldrich, MO, USA) for precipitation of RNA followed by ethanol (100% and 70%) (HiMedia, Mumbai, India) wash to remove residues were used in the procedure. DNase I (New England Biolabs, MA, USA) and 10X DNase I buffer along with the enzyme were used to remove DNA.

RNA isolation

In this experiment, four methods were used for RNA isolation viz., CTAB-phenol/chloroform method [23], NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany), modified CTAB-chloroform/isoamyl alcohol method [29] and optimized CTAB-LiCl-sodium acetate method. Extraction procedures are briefly explained and the optimized protocol is detailed in the following sections.

Method 1: CTAB-phenol/chloroform-based RNA extraction

RNA extraction was performed according to Rubio-Piña and Zapata-Pérez [23]. In this method, around 200 mg root tissue was crushed with liquid nitrogen and incubated for 10 min at 65°C in CTAB extraction buffer along with β-mercaptoethanol (HiMedia, Mumbai, India). This was followed by chloroform, phenol-chloroform (1:1) and chloroform-isoamyl alcohol (24:1) based separation of RNA. After centrifugation, the supernatant was collected and precipitated using LiCl (8 M) (Sigma-Aldrich, MO, USA). The precipitated RNA was subjected to DNase I (New England Biolabs, MA, USA) treatment to remove DNA contamination. Additional phenol-chloroform extraction was carried out to precipitate RNA after DNase I treatment. RNA was precipitated using sodium acetate (3 M) (Sigma-Aldrich, MO, USA) and washed with ethanol to remove excess reagents. The final pellet was diluted in 50 μl nuclease-free water.

Method 2: NucleoSpin RNA Plant & Fungi kit

In this method, a commercial NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) was used. RNA isolation was done in accordance with the manufacturer's protocol. Briefly, the plant tissue was ground in liquid nitrogen and added to the lysis buffer. The mixture was centrifuged and the supernatant was filtered through the NucleoSpin RNA Plant and Fungi filter column to remove plant debris. Binding buffer was added to the lysate obtained and loaded onto the NucleoSpin RNA Plant and Fungi column. The method uses silica-membrane filtration of the lysate-binding buffer, where the RNA is adsorbed into the silica membrane. The lysis buffer contains guanidine hydrochloride while the binding solution contains LiCl. Further washing steps with two different wash buffers were conducted to remove reagents and contaminating cellular components. The RNA was finally eluted from the membrane with 50 μl RNase-free water.

Method 3: extraction without phenol

The modified method of Kiefer et al. [29] uses a CTAB-based extraction buffer (with spermidine removed) containing β-mercaptoethanol (HiMedia, Mumbai, India) and isopropanol (Sigma-Aldrich, MO, USA) precipitation. Proteinase K (HiMedia, Mumbai, India) was added as an additional step. Briefly, powdered mangrove root tissues were added to a prewarmed extraction buffer at 65°C. This was followed by chloroform-isoamyl alcohol separation of RNA. Chloroform-isoamyl alcohol was again added to the supernatant to increase RNA purity. The RNA was precipitated from the supernatant using isopropanol (Sigma-Aldrich, MO, USA). The pellet was treated with DNase I (New England Biolabs, MA, USA) and precipitation with isopropanol (Sigma-Aldrich, MO, USA) and ethanol wash was performed to remove contaminants. The final RNA pellet was dissolved in 50 μl nuclease-free water.

Method 4: modified method using CTAB & LiCl

The standardized method follows CTAB (HiMedia, Mumbai, India) and LiCl (Sigma-Aldrich, MO, USA) precipitation of RNA. Using a mortar and pestle (baked at 250°C for 2 h), about 100–200 mg of mangrove root was ground into a fine powder using liquid nitrogen. The extraction buffer was preheated at 65°C for 15 min. The ground sample was then transferred to a 2 ml microcentrifuge tube containing 0.8 ml extraction buffer and 0.1 ml β-mercaptoethanol (HiMedia, Mumbai, India). Further, 100 μl proteinase K (HiMedia, Mumbai, India) and 500 μl chloroform-isoamyl alcohol (24:1) was added. The solution was then centrifuged at 13,000 r.p.m. for 5 min at 4°C. All centrifugation steps were performed at 4°C. The resultant supernatant was transferred to a 2 ml microcentrifuge tube, and 250 μl of chloroform-isoamyl alcohol (24:1) was added. The tubes were then vortexed for a few minutes and centrifuged at 13,000 r.p.m. for 2 min. The supernatant was then transferred to a 1.5 ml microcentrifuge tube, and LiCl (Sigma-Aldrich, MO, USA) was added to double the supernatant's volume. Tubes were kept for incubation overnight at -20°C. The protocol then proceeded with centrifugation at 13,000 r.p.m. for 5 min. The resultant pellet was then dissolved in 89 μl H₂O, 10 μl DNase I reaction buffer (10X) and 1 μl DNase I (New England Biolabs, MA, USA). The solution was then incubated at 37°C for 10 min, and 1 μl 0.5 M EDTA (HiMedia, Mumbai, India) was added; 10 μl sodium acetate (Sigma-Aldrich, MO, USA) and 250 μl 100% ethanol (HiMedia, Mumbai, India) were added next. The tube was vortexed and kept for incubation at -20°C for 3 h, followed by centrifugation at 13,000 r.p.m. for 5 min; 1 ml of 70% cold ethanol (HiMedia, Mumbai, India) was added and centrifuged at 13,000 r.p.m. for 5 min. The supernatant was decanted, and the pellet was air dried for 10–20 min at room temperature (16–18°C); 50 μl of RNase-free water was used to dilute RNA. Diluting in 25 μl nuclease-free water gives a higher concentration per μl. Figure 2 shows a graphical representation of the optimized method reported in this study.

RNA quality assessment

RNA yield and purity were analyzed using a NanoDrop One UV-Vis Spectrophotometer (Thermo Fisher Scientific, MA, USA). A260/A280 and A260/A230 ratios were assessed to determine purity. The integrity was visualized through denaturing gel electrophoresis; 1.2% MOPS-formaldehyde-agarose gel was stained in ethidium bromide (HiMedia, Mumbai, India). Total RNA concentrations from the standardized protocol and commercial kit were assessed using a Qubit 3.0 fluorometer (Invitrogen, CA, USA, Thermo Fisher Scientific, MA, USA) at AgriGenome Labs Pvt Ltd., Kerala, India. RNA integrity number (RIN) values for the same were measured using a Tapestation 2200 (Agilent technologies, CA, USA) at AgriGenome Labs Pvt Ltd., Kerala, India. RIN is the measurement of RNA quality provided by the Tapestation 2200 (Agilent) based on electrophoretic separation and fluorescence detection. Lower RIN values point to a higher degradation rate of RNA samples [45].

cDNA synthesis, next-generation sequencing & qRT-PCR

cDNA for RT-PCR reactions were prepared using the iScript cDNA synthesis kit (BioRad, CA, USA). RNA isolated from K. candel and R. mucronata was used with a NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) and the modified protocol, respectively. Gene-specific primer sequences used for RT-qPCR are listed in Table 1. RT-qPCR was performed at 95°C for 2 min, 40 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 15 s with a reaction volume of 10 μl (5 μl TB Green Premix Ex Taq II (Takara, Shiga, Japan); 1 μl forward primer; 1 μl reverse primer, 100 ng cDNA and nuclease-free water) using the Lightcycler 480 (Roche, Rotkreuz, Switzerland). Isolated RNA with RIN values >7 was used for downstream applications such as transcriptome sequencing. Poly (A) capture of mRNAs was followed by cDNA library preparation and sequenced using NovaSeq 6000 (Illumina, CA, USA) at AgriGenome Labs Pvt Ltd., Kerala, India.

Results & discussion

Isolation of RNA from mangroves is challenging as they contain large amounts of polyphenols and polysaccharides, which precipitate along with nucleic acids, affecting yield and purity. Several kits are available for RNA isolation but are not always successful in obtaining good-quality RNA owing to the presence of specialized metabolites. The metabolite composition varies among plant species; thus, different approaches are utilized to obtain good-quality RNA [32,46].

In the present study, four methods were used to isolate RNA from R. mucronata and K. candel root tissues, including the CTAB-phenol/chloroform-based protocol (method 1), NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) (method 2), protocol without phenol (method 3) and standardized method (method 4; Table 2 & Figure 3). Routinely used protocols on mangroves produced unsuitable results, leading to the use of commercial kits. Among the commercial kits, conventionally used kits such as TRIzol (Invitrogen, CA, USA) and RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) failed to extract good-quality RNA from K. candel and R. mucronata. TRIzol (Invitrogen, CA, USA) is a phenol-guanidinium isothiocyanate-based reagent. On the other hand, the RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) is based on guanidinium isothiocyanate and silica membrane filtration with no LiCl or ethanol precipitation. Neither is suitable for the extraction of RNA from K. candel and R. mucronata roots. In contrast, the NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany) uses guanidine hydrochloride in the lysis buffer and LiCl in the binding buffer, thus being fruitful in isolating RNA from K. candel. The NucleoSpin RNA Plant and Fungi isolation kit (Macherey-Nagel, Düren, Germany) extracted RNA from K. candel (A260/A280: 2.10) but was unsuccessful in isolating good-quality RNA from R. mucronata (A260/A280: 1.51). The RNA isolation method by Kiefer et al. [29] uses chloroform/isoamyl alcohol separation and was used on mangrove tissues, and also extracted low-quality RNA, although the absorbance ratios were higher (A260/A280: 1.9–2.04; A260/A230: 1.15–2.26). Thus, an optimized protocol was developed to isolate good-quality RNA from R. mucronata and K. candel.

Method 1: CTAB-phenol/chloroform-based RNA extraction

First, a procedure successful in mangrove plants based on phenol-chloroform extraction yielded a low concentration of total RNA in both K. candel (∼14.35 μg/g FW) and R. mucronata (∼5.08 μg/g FW) compared with other methods studied. A260/A280 and A260/A230 ratios obtained for both plant species using method 1 are shown in Table 2. An A260/A280 ratio of ∼2 indicates pure RNA, while ratios that deviate from this value can be due to a low concentration of RNA or the presence of residual reagents [47]. A A260/230 ratio within the range of 2.0–2.2 is considered pure RNA and lower values indicate the presence of contaminants that absorb at a wavelength of 230 nm [47,48]. Rubio-Piña and Zapata-Pérez [23] have isolated RNA from Languncularia racemosa and Avicennia germinans. Since the metabolic content varies with each species [48], method 1 may not be suitable for separating and precipitating RNA from K. candel and R. mucronata.

Method 2: NucleoSpin RNA Plant & Fungi kit

The second method used was a commercially available plant RNA isolation kit (NucleoSpin RNA Plant and Fungi kit, Macherey-Nagel, Düren, Germany). The silica membrane filter was efficient in removing cellular debris, metabolites and salt and isolating good-quality RNA from K. candel (Figure 3). Method 2 was able to isolate RNA from K. candel with a concentration of ∼81.39 μg/g FW, A260/A280 ratio of 2.1 and A260/A230 ratio of 2 (Table 2). RIN values of 9.9 indicate that the commercial kit-based method 2 successfully isolates highly intact RNA from K. candel roots (Figure 4). Electropherograms of total RNA extracted from K. candel are illustrated in Figure 4. The high-quality RNA thus obtained can be used for further molecular studies involving cDNA library preparation, transcriptome sequencing and gene expression [14,49] in mangroves. However, the yield (∼48.925 μg/g FW) and purity (A260/A280: 1.51; A260/A230: 0.52) of RNA obtained from R. mucronata root were lower than the acceptable range. Similarly, CTAB-phenol-chloroform-based extraction (method 1) showed lower absorbance ratios and less concentration of RNA compared with other procedures (Table 2). These results suggest that phenol-chloroform extraction of RNA may not be efficient for mangrove plant root samples. Poor absorption ratios may be associated with improper extraction and residual contaminants or nucleic acids [48]. Further optimization of the isolation protocol is needed for R. mucronata since silica membrane filtration may be unsuitable due to high polyphenol and polysaccharide content. Oxidized products of polyphenols and polysaccharides can bind with RNA and elute along with nucleic acids [49].

Method 3: extraction without phenol

Method 3, which is based on CTAB and chloroform-isopropanol precipitation, could not isolate high-purity RNA from the roots of either mangrove species. However, an average concentration of 46.40 μg/g FW and better yield were observed for K. candel (Table 2). This may indicate interference due to specialized metabolites, proteins or carbohydrates in isolated RNA. RNA obtained from K. candel using the modified method 3 had absorbance ratios of A260/A280 and A260/230 close to 1.99 and 2.26, respectively, whereas, in R. mucronata, the absorbance ratios were 2.04 and 1.15, respectively. But when visualized in 1.2% formaldehyde-agarose gel, the bands appeared smeared (Figure 3). Smeared RNA indicates degradation while two distinct bands (28S and 18S) indicate intact RNA [50]. Hence, this method might not be suitable for isolating RNA from K. candel or R. mucronata and needs further optimization.

Method 4: modified protocol using CTAB & LiCl

Several studies have isolated RNA from recalcitrant tissues such as mangrove leaves and roots [23,30,51]. The use of phenol-chloroform-based protocols was not suitable for RNA isolation from K. candel and R. mucronta. The modified method reported in this study isolated good-quality RNA from both K. candel and R. mucronata (Figure 3). The concentration of RNA obtained was analyzed using absorbance values at a wavelength of 260 nm. A260/A280 and A260/230 ratios of RNA isolated from R. mucronata using the modified method were 2.04 and 2.12, respectively, with an average concentration of 46.93 μg/g FW. The RIN value for RNA isolated from R. mucronata was 9.6, indicating highly intact RNA was obtained with a modified CTAB-LiCl protocol (Figure 4). Interestingly, absorbance ratios for K. candel were also higher. The A260/A280 and A260/230 absorbance ratios were 2.14 and 2.21, respectively. Thus, this modified protocol isolated good-quality RNA from K. candel and R. mucronata.

High-quality RNA was obtained using modified protocol

Analysis of isolated RNA using agarose gel electrophoresis showed that method 4 had an intense band for both mangrove species with less degradation (Figure 3). Distinct bands suggest that the isolated RNA was not degraded, while method 3 had bands with degradation. Clear and intense bands of RNA isolated from K. candel were observed with method 2. However, for the R. mucronata samples isolated with method 2, the bands were not clear. Indistinct or smeared bands suggest RNA degradation [50], and faint bands were observed for RNA isolated from K. candel using method 1 (Figure 3). These results suggest that the modified protocol and NucleoSpin RNA Plant and Fungi isolation kit (Macherey-Nagel, Düren, Germany) are efficient in isolating good-quality RNA from mangrove root tissues containing a high level of polyphenols. This again emphasizes the importance of removing phenol in RNA isolation to reduce contamination.

Total RNA isolated with the NucleoSpin column was comparatively higher (81.39 μg/g FW) for K. candel. In the case of R. mucronata, the modified CTAB-LiCl protocol isolated a higher yield of RNA than the kit-based method. Using CTAB (HiMedia, Mumbai, India) in an extraction buffer helps precipitation of polysaccharides [36]. In the modified protocol, the addition of proteinase K (HiMedia, Mumbai, India) to the extraction buffer-sample mix is responsible for the degradation of RNases [29,52]. These steps are crucial for the removal of protein and polysaccharide contaminants before the separation of nucleic acids. Compared with the previously published protocol by Rubio-Piña and Zapata-Pérez [23], chloroform-isoamyl alcohol was used to separate RNA to the aqueous phase from contaminants such as phenol, protein and polysaccharide residues [26]. Phenol-chloroform precipitation was used by Rubio-Piña and Zapata-Pérez [23], which can increase the risk of phenol contamination. Moreover, the same method uses phenol-chloroform steps again after the DNase treatment. But, the optimized protocol used only chloroform (Sigma-Aldrich, MO, USA) and isoamyl alcohol (HiMedia, Mumbai, India) to separate RNA and further precipitation with LiCl (Sigma-Aldrich, MO, USA). LiCl binds with RNA and forms a precipitate [36]. LiCl is used in CTAB-phenol/chloroform, NucleoSpin RNA Plant and Fungi isolation kit and the current modified method. In the modified method, the LiCl (Sigma-Aldrich, MO, USA) precipitation was followed by DNase I (New England Biolabs, MA, USA) treatment, precipitation using sodium acetate (Sigma-Aldrich, MO, USA) and ethanol (HiMedia, Mumbai, India) washes isolated high-quality RNA [43]. At the same time, in method 1, DNase treatment was followed by sodium acetate precipitation and additional separation/precipitation steps using a phenol-chloroform reagent. This might have compromised the RNA integrity. Although the optimized protocol is time-consuming, the extracted RNA has a high yield and integrity crucial for RNA-based molecular studies.

High-integrity RNA is crucial for library preparation intended for transcriptome sequencing [53]. RIN values of 7.5–10, with 10 representing the lowest degradation in samples, are considered acceptable before proceeding with cDNA library preparation [53]. Electropherograms generated from the Tapestation 2200 (Agilent) showed distinct peaks for 18S and 28S ribosomal RNAs for K. candel and R. mucronata (Figure 4A). RIN values for K. candel RNA isolated with method 2 ranged from 7.6 to 9.9. In the case of R. mucronata total RNA isolated with the modified protocol, RIN values ranged from 7.5 to 9.6. Poly (A) enrichment selectively separates mRNA fraction [53] and can be useful for targeted studies on mRNA expression analysis. The Tapestation profile of the cDNA library prepared also shows that a good-quality library was obtained with no presence of additional adapters and primer dimers (Figure 4B) [54]. cDNA synthesis was followed by sequencing in the Illumina platform. The quality of raw reads was assessed based on the Phred score. Higher Phred scores indicate better base calling accuracy [55]. Phred quality scores (Q score) >30 obtained for the samples indicate 99.9% accuracy of the sequencing platform. More than 90% of the reads generated from K. candel and R. mucronata had a Q score >30 (Figure 4C). RT-qPCR was performed after the synthesis of cDNA from total RNA obtained using the optimized protocol. Three genes from K. candel (Tubulin, Gpn2 and Kanadaptin) and R. mucronata (Actin, PGK and STK) were selected for PCR amplification (Figures 5 & 6). Tubulin and Actin were the reference genes for K. candel and R. mucronata, respectively. The amplification curve plot indicates that the Ct value was within the accepted range, and a single melt curve peak for all genes suggests the specificity of the primers (Figures 5A–C & 6A–C). Visualization of RT-qPCR products in 4% agarose gel electrophoresis showed clear bands (Figures 5D & 6D for K. candel and R. mucronata, respectively). Further research based on transcriptome sequencing data is currently ongoing in the authors' lab.

Conclusion

Isolation of RNA from plants containing high phenolics, polysaccharides and carbohydrates is challenging as these compounds interfere with the extraction procedure and thereby decrease its purity and integrity. Elimination of secondary metabolites, DNA contamination and reduction of interfering reagent residues present in the sample can produce high-quality total RNA. Obtaining good-quality total RNA from mangroves is necessary for downstream applications including studying molecular mechanisms underlying mangrove adaptation to environmental stresses. In the present study, an RNA isolation method was developed to obtain high-purity RNA without the contamination and inference of other compounds. The essential steps in this protocol to obtain high-quality RNA include the use of β-mercaptoethanol to inhibit RNase activity, proteinase K to degrade proteins and LiCl and sodium acetate to precipitate RNA. DNA contamination is removed by DNA degrading enzymes such as DNase I to get RNA with high yield and purity. The standardized protocol and Nucleospin Plant and Fungi RNA isolation kit were able to isolate suitable quality total RNA from R. mucronata and K. candel, respectively. Moreover, agarose gel electrophoresis showed dark and intact bands for these methods and the RIN values ranged 7.5–9.9. Hence, the optimized protocol was successful in obtaining high-quality RNA and can be used for downstream applications, including cDNA synthesis, gene library construction, gene amplification, transcriptome analysis and qRT-PCR.

Future perspective

This modified method successfully obtained high-quality RNA from mangroves. Considering the lack of data on mangrove stress biology, future studies focused on the molecular biology of mangroves should put effort into resource building and can utilize the proposed method for isolating RNA from other less-studied species. Moreover, the modified method reported in this study can isolate RNA from problematic plant species with high concentrations of polysaccharides, polyphenols and other secondary metabolites. Such efforts will aid in identifying molecular mechanisms behind the growth, development and stress tolerance of diverse plant species.