Extractions of High Quality RNA from the Seeds of Jerusalem Artichoke and Other Plant Species with High Levels of Starch and Lipid

Jerusalem artichoke (Helianthus tuberosus L.) is an important tuber crop. However, Jerusalem artichoke seeds contain high levels of starch and lipid, making the extraction of high-quality RNA extremely difficult and the gene expression analysis challenging. This study was aimed to improve existing methods for extracting total RNA from Jerusalem artichoke dry seeds and to assess the applicability of the improved method in other plant species. Five RNA extraction methods were evaluated on Jerusalem artichoke seeds and two were modified. One modified method with the significant improvement was applied to assay seeds of diverse Jerusalem artichoke accessions, sunflower, rice, maize, peanut and marigold. The effectiveness of the improved method to extract total RNA from seeds was assessed using qPCR analysis of four selected genes. The improved method of Ma and Yang (2011) yielded a maximum RNA solubility and removed most interfering substances. The improved protocol generated 29 to 41 µg RNA/30 mg fresh weight. An A260/A280 ratio of 1.79 to 2.22 showed their RNA purity. Extracted RNA was effective for downstream applications such as first-stranded cDNA synthesis, cDNA cloning and qPCR. The improved method was also effective to extract total RNA from seeds of sunflower, rice, maize and peanut that are rich in polyphenols, lipids and polysaccharides.


Results and Discussion
Our initial attempt using the five existing methods to extract total RNA from Jerusalem artichoke dry seeds yielded variable, unsatisfactory results (Table 1 and Figure 1). The resulting yields of total RNA were unexpectedly low, smaller than 15 µg RNA/30 mg fresh weight. Clearly, the efforts with the plant RNeasy ® method (Qaigen, Hilden, Germany) and TRIzol ® reagent (Invitrogen, Carlsbad, CA., Commercial RNA isolation kits were not designed for use with plant tissues containing high concentrations of polyphenols, polysaccharides and other secondary metabolites. There are reports modifying extraction buffers in the plant RNeasy ® mini kit and leading to successful RNA extraction on leaves and bud tissue of grapevine and from leaves, bud and cane tissues of woody plant [15,16], but this has rarely been tested on seeds. Applying the published protocols developed for RNA isolation from cereal seeds [3,7] and sunflower dry seeds [6] generated poor quality and low yield RNA from Jerusalem artichoke dry seeds. We also observed that the pellet became viscous when phenol and chloroform were added. The method of Wang et al. [7] was able to extract 9 and 13 µg RNA/30 mg fresh weight from Jerusalem artichoke cv. JA37 and HEL53, respectively. A260/A280 were 1.82 and 1.77 while A260/A230 ratios of total RNA were 1.56 and 1.55 (Table 1), suggesting their impurity.
Considering the high levels of starch and lipid present in Jerusalem artichoke seeds, we modified the methods of Li and Trick [3] and Ma and Yang [6] by adjusting the amounts of reagents and adding extra important steps. Specifically, the original Li and Trick [3] method was modified as MLT method by adding a step containing TRIzol ® reagent which contains phenol and guanidine isothiocyanate, a strong denaturant. This solution helps to remove all traces of interfering protein and other metabolites [17]. The addition of 750 µL instead of 400 µL of extraction buffer and using 500 µL of TRIzol ® reagent instead of 250 µL of extraction buffer II in Li and Trick [3] protocol helped to get 16 and 15 µg RNA/30 mg fresh weight from seeds of Jerusalem artichoke cv. JA37 and HEL53 within approximately one and a half hours (Table 1; Figure 1) which is higher than the amount of RNA extracted from more seed weight (60 mg) using the original protocol of Li and Trick [3] (Table 1). These efforts generated successful extractions of total RNA from Jerusalem artichoke dry seeds, as illustrated in Table 1 and Figure 1. The yields of total RNA by MLT were significantly higher than those obtained using the other unmodified methods.
The original method of Ma and Yang [6] utilized high salt concentration (8 M LiCl) and β-mercaptoethanol in the extraction buffer to inactivate RNase activity. The extraction and solubilization buffers, in the presence of PVP and SDS in two-steps RNA isolation of Ma and Yang [6] helped to remove polysaccharides, proteins and polyphenolic compounds [17]. PVP also prevented the oxidation of polyphenols, which occur after long air exposure of samples. The polyphenol removal was carried out using two repeat chloroform extractions prior to RNA extraction using Trizol ® . We improved the original protocol further as MMY method with several considerations. These included (1) the reduction of seed sample to 30 mg; (2) the change in the temperature and incubation time to room temperature for 5 min; (3) purification of a pellet instead of the suspension in the original protocol of Ma and Yang [6] after adding chloroform to the mixture of fine powder, the extraction buffer and β-mercaptoethanol, and ethanol; LiCl precipitated RNA as a pellet, leaving DNA in the supernanant; (4) the replacement of NaAc-saturated acidic phenol and chloroform by Trizol ® reagent and chloroform; and (5) precipitation of total RNA using isopropanol instead of ethanol and 3 M NaAc, pH 5.2. These modifications helped to overcome the problem of viscous pellets in the original method of Trizol ® and produced significant higher yields of total RNA (35 µg RNA/30 mg) than those using the original method of Ma and Yang [6] and also the MLT method (Table 1; Figure 1). The MMY method also made the RNA extraction effort simpler and more reproducible and the overall effort required approximately one and a half hours. The quality of RNA prepared by this method was demonstrated by intact sharp 28S and 18S rRNA bands and no sign of RNA degradation on agarose gel. The extracted RNA was of high quality indicated by the A260/A280 and the A260/A230 ratios ( Figure 1 and Table 1) [18,19].
To assess the efficiency of the MMY method, we further assayed another set of dry seeds from 10 extra JA accessions of diverse origins (listed in Table 2). The results are encouraging and proved to be applicable for all Jerusalem artichoke seeds independent of their mother plants ( Figure 2 and Table 3).   The yields ranged from 29 to 41 µg RNA/30 mg fresh weight. These results helped to demonstrate the advantage of the improved method in rapid isolation of RNA from small amounts compared to other seed RNA extraction protocols [3,6,7,[20][21][22][23]; the latter mostly used 50-100 mg seed material.
To evaluate the integrity of the extracted RNA using the MMY method, qPCRs were used to amplify a housekeeping gene (HtEF1-alpha) and genes (HtGA2-oxidase, HtGA20-oxidase and HtPHYB), which are known to play specific roles in the induction of seed germination in Arabidopsis [24,25]. The synthesis of cDNAs using the extracted RNA and reverse transcriptase enzyme was successful. The target bands with expected sizes of the HtEF1-alpha, HtGA2-oxidase, HtGA20-oxidase and HtPHYB genes were observed ( Figure 3). The qPCR cycle thresholds (Ct) was between 18 and 40 cycles, and the melting curve was specific, with a single peak occurring at about 84 °C, 82 °C, 82 °C and 82 °C for HtEF1-alpha, HtGA2-oxidase, HtGA20-oxidase and HtPHYB, respectively ( Figure 3). These results indicated that the MMY method was effective in extracting total RNA for the transcript analysis of gene expression in Jerusalem artichoke seeds. We also assessed the applicability of the MMY method to extract total RNA from other plant species with high levels of starch and lipid. Specifically, we isolated total RNA from the seeds of the  Table 2). The initial assessment revealed that genomic DNA was still contaminated in all plant species studied except Jerusalem artichoke. Thus, the genomic DNA was removed by DNaseI digestion following phenol/chloroform purification. The RNA was then precipitated. Figure 4 illustrates the absence of undesirable contaminants. The yield of total RNA isolated from these seeds ranged from 25-63 μg/30 mg fresh weight (Table 3). RNA absorbance (A260/280 and A260/230) values of sunflower and marigold are below 1.8 showing the impurity of protein ( Table 3). The Ma and Yang [6] original protocol revealed low purity (A260/280 was 1.50) and low yield of total RNA (1.71 μg/100 mg) from dry sunflower seeds when 350 µL of ethanol was added to the extraction buffer. The total RNA extracted here is much higher than that of Ma and Yang [6] original protocol. As illustrated in Figure 4, the MMY method produced large amounts of total RNA from rice, maize and peanut seeds (Figure 4 and Table 3). To further evaluate the integrity of the total RNA extracted from these assayed species, SRAP-cDNA amplifications and a transcript analysis of EF1-alpha gene was performed. Figure 5 showed the gene expression was successfully detected in the seeds of peanut, maize, sunflower, and rice. However, the RNAs extracted from marigold were not adequate in these downstream analyses. Clearly, the improved method was effective in extracting RNA of desired quality and quantity from small amount of seeds from several other plant species studied here.

Plant Material
This study assayed 18 seed samples of Jerusalem artichoke, sunflower, maize, rice, peanut and marigold ( Table 2) Chia Tai Co. Ltd. and Chua Yong Seng Seed Co. Ltd., respectively. The Jasmine rice cultivar was obtained from Khon Kaen Rice Research Center.

RNA Extraction
Total RNA was initially extracted from 30 mg ground seed material with five independent replications using five existing extraction methods. The methods are (1) TRIzol ® reagent (Invitrogen), (2) Plant RNeasy mini kit (Qaigen, Germany), (3) method of Wang et al. [7], (4) method of Li and Trick [3], and (5) method of Ma and Yang [6]. These initial efforts generated an unsatisfactory amount and purity of total RNA, and further efforts were made to improve the methods of Li and Trick [3] and Ma and Yang [6]. Before operation, all plastic materials were autoclaved. Glass material and the mortar and pestle were baked for 3-6 h at 180 °C.

MLT Method
(1) Thirty mg seeds were ground with mortar and pestle in the presence of liquid nitrogen. Transfer immediately into 1.5 mL microcentrifuge tubes.
(2) A 750-µL extraction buffer was added and mixed by vortex.
(3) The high molecular weight impurities and lipids were removed by adding 750 µL of phenol: chloroform (1:1), mixed gently and centrifuged at 12,000 rpm for 10 min.
(4) The supernatant was collected and the phenol-chloroform extraction was repeated. The supernatant was transferred to a new RNase-free tube.
(5) Supernatant was added with an equal volume (about 500 µL) of TRIzol ® reagent, mixed thoroughly and incubated at room temperature for 10 min.
(6) An equal volume of chloroform: isoamyl alcohol (24:1) was added to the tube, mixed gently and centrifuged at 12,000 rpm for 10 min.
(7) The supernatant was transferred to a new RNase-free tube. One volume of isopropanol (about 500 µL) and 200 µL of 1.2 M NaCl were added and mixed by inversion several times, and incubated at −20°C for 15 min. The pellet was precipitated by centrifugation at 10,000 rpm for 10 min. The supernatants were discarded and the RNA pellets were washed carefully with 70% ethanol. (8) The RNA pellets were re-suspended in 30-µL RNase-free water and stored at −70 °C. The modified protocol MLT requires a lower amount of seed (30 mg vs. 50-100 mg), more extraction buffer (750-µL vs. 400-µL), a repeat of the phenol-chloroform extraction (750-µL vs. 250-µL ) and replace 70% guanidinium sulfate (w/v), 0.75 M sodium citrate, 10% laurylsarcosine, 2 M sodium acetate, pH 4.0 with TRIzol ® reagent.

MMY Method
(1) Thirty mg seed was ground with mortar and pestle in the presence of liquid nitrogen and PVP40.
(2) Transfer immediately into 1.5 mL centrifuge tubes. Then, 0.9 mL of the extraction buffer and 350 µL of ethanol were added, mixed by vortex, and incubated at room temperature for 5 min.
(3) The high molecular weight impurities and lipids were removed by adding 100 µL chloroform mixed gently and centrifuged at 5,000 rpm for 3 min.
(4) The supernatant was discarded. The pellet was dissolved in 550 µL solubilization buffer. The chloroform extraction was repeated with equal volume of solubilization buffer and centrifuged at 5,000 rpm for 3 min.
(5) The supernatant was transferred to a new RNase-free tube and added with 500 µL of TRIzol ® reagent and 100 µL of chloroform, mixed thoroughly, incubated at room temperature for 2 min and centrifuged at 12,000 rpm for 10 min.
(6) The aqueous phase was transferred to a new RNase free tube. An equal volume of isopropanol was added and incubated at −20 °C for 15 min. The pellet was precipitated by centrifugation at 12,000 rpm for 10 min. The supernatant was discarded and the pellet was allowed to dry, followed by re-suspending the pellet in 30 µL RNase-free water.
The MMY protocol requires a lower amount of seed (30 mg vs. 60 mg), more ethanol (350-µL vs. 250-µL) and a much shorter incubation time after adding extraction buffer (5 min at room temperature instead of overnight at 4 °C). The purification of pellet was made instead of the suspension in the original protocol of Ma and Yang [6] after adding chloroform to the mixture of fine powder, the extraction buffer and β-mercaptoethanol. Also, the supernatant was added with 500 µL of TRIzol ® reagent and 100 µL of chloroform instead of using 500 µL NaAc-saturated acidic phenol and 300 µL chloroform in the second round of purification.

RNA Extraction from Seeds of Other Plant Species
RNA extraction from seeds of other plant species was done according to the MMY method, as described above. After the RNA precipitation with isopropanol, the pellet was dissolved in 26 µL of RNase-free water and added with 3 µL buffer 10× buffer and 1 µL (10 units) of RQ1 RNase-Free DNase (Promega). Incubate 30 min at 37 °C, followed by adding the RNase-free water (470 µL). The phenol: chloroform (1:1) extraction was repeated with equal volumes (about 500 µL) and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a new RNase-free tube, followed by adding an equal volume of isopropanol and 200 µL of 1.2 M NaCl. Incubate at −20 °C for 15 min. The pellet was precipitated by centrifugation at 12,000 rpm for 15 min. The supernatant was discarded and the pellet was allowed to dry followed by re-suspended the pellet in 30 µL RNase-free water.

RNA Evaluation
The concentration and quality of total RNA extracted from all five existing methods were analyzed based on the 260 nm/280 nm and 260/230 absorbance ratios. An aliquot of total RNA was used in the Agilent 8453 UV-visible spectrophotometer according to manufacturer's instructions. The yield of total RNA extracted from 30 mg fresh weight was reported. The variation in the efficiency of RNA extractions was analyzed using Statistix 8 [27]. RNA integrity was evaluated from the 28S and 18S rRNA bands from 5 µL of total RNA on 1.0% agarose gel electrophoresis. The gels were stained with ethidium bromide and visualized with UV light. The photographs were taken using Vilber Lourmat (France) and the images were inverted in Adobe Photoshop. The evaluations were initially performed on the total RNA using the five extraction methods, followed by those using the MMY method from the 18 seed samples representing Jerusalem artichoke, sunflower, maize, rice, peanut and marigold.

Reverse Transcription and Subsequent Application
Transcript expression of selected genes was quantified by qPCR. After DNase treatment using RQ1 RNase-Free DNase (Promega), five replicates of 1.25 µg of total RNA from Jerusalem artichoke seeds were reverse-transcribed in a 10 µL reaction using OligodT (2.5 µM) and digested with RNaseH according to the SuperScript™ Vilo kit instructions (Invitrogen). After the first strand cDNA synthesis, the solution was 10× diluted to a final concentration of 125 ng/µL. PCR amplification was made based on 2 µL of diluted 1st strand-cDNA using Elongation factor 1-alpha (HtEF1-alpha), HtGA2-oxidase, HtGA20-oxidase, and Phytochrome B (HtPHYB) gene-specific oligonucleotide primers (Table 4). These primers were designed based on gene homologies from the Compositae Genome Project Database [28] which were confirmed using BLAST. The qPCR experiments were performed in a thermocycler called "LightCycler ® 480" (Roche). The reaction was conducted with a final volume of 20 µL using the LightCycler ® 480 SYBR Green I Master kit (Roche) according to the manufacturer's instructions. The qPCR condition was as follows: at 94 °C for 5 min; 45 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 min, with a final extension at 72 °C for 5 min. The PCR products were also analyzed on 1.0% agarose electrophoresis and visualized as described above. The gene expression analysis was done based on the total RNA extracted from seeds of Jerusalem artichoke and other assayed plant species.

Conclusions
We report an improved method MMY for high-quality and -quantity RNA extraction from dry seeds of Jerusalem artichoke with high levels of starch and lipid. The improved method is also applicable for seeds of sunflower, rice, maize and peanut, which are rich in polyphenols, lipids and polysaccharides. The effectiveness of the improved method to extract total RNA from seeds of these assayed plant species was confirmed with qPCR analysis of selected genes.