An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.


Results
RNA samples with higher quality and yield were extracted by the modified TRIzol method from root, stem and leaf in L. davidii var. unicolor. Three methods were used for extracting RNA samples from root, stem and leaf tissues of L. davidii var. unicolor, including the modified TRIzol method, Kit method and CTAB method. In Lilium root, RNA samples obtained from the modified TRIzol method showed that the A260/A230 and A260/A280 ratios were closed to 1.5 and 2.1, and RNA samples gained from the CTAB method showed higher ratios (ratios of A260/A230 and A260/A280: 1.9 and 2.2, respectively) ( Table 1). However, RNA prepared from root with the Kit method showed lower absorbance ratios (ratios of A260/A230 and A260/A280: 1.0 and 2.0, respectively). Among these methods, the highest RNA concentration was obtained by using the modified TRIzol method (565.39 ng μL −1 ) ( Table 1). For stem tissue of Lilium as shown in Table 1, the A260/A280 and A260/A230 ratios of the RNA samples extracted by the Kit and CTAB methods were less than 1.0 and 1.8, respectively, indicating that the extracted RNA was of low purity. However, the modified TRIzol method resulted in higher A260/A230 ratio (1.0), A260/A280 ratio (1.97) and yield (165.69 ng μL −1 ). Therefore, high-quality total RNA could be isolated from Lilium stem by using the modified TRIzol method. When RNA was extracted from Lilium leaf using the Kit and CTAB methods, the spectrophotometric results showed that the A260/A230 ratios of RNA ranged from 0.91 to 1.07, and the A260/A280 ratios ranged from 1.69 to 2.01, indicating that the RNA samples were contaminated by proteins, polyphenols and polysaccharides. However, when using the modified TRIzol method, the A260/A230 and A260/A280 ratios and the RNA concentrations were increased significantly (Table 1).
In order to compare TRIzol method and the modified TRIzol method, the RNA samples obtained from root, stem and leaf by use of the TRIzol method were also analyzed. The A260/A230 and A260/A280 ratios of the RNA gained by the modified TRIzol method showed no difference with those of the TRIzol method. Nevertheless, the RNA concentrations of root and stem obtained from the TRIzol method (394.98 ng μL −1 and 133.99 ng μL −1 , respectively) were significantly lower than that from the modified TRIzol method (Table 1). Taken together, the modified TRIzol method is the most efficient and reproducible method to extract high-quality total RNA from root, stem and leaf tissues of L. davidii var. unicolor.
The integrity of isolated RNA samples was then tested by using 1% agarose gel electrophoresis. The RNA samples from Lilium root isolated by the TRIzol method showed obviously degradation, and the detected bands of 28S and 18S ribosomal RNA (rRNA) from stem and leaf were quite shallow (Fig. 1A). Moreover, the RNA samples gained from the modified TRIzol and Kit methods showed legible and integrated bands of 28S rRNA and 18S rRNA without obvious DNA contamination (Fig. 1B,C). However, the RNA samples obtained by the CTAB method showed visible chromosomal DNA bands, and the corresponding RNA bands were degraded seriously (Fig. 1D). To this end, the obtained total RNA by the modified TRIzol method showed high yield and quality as revealed by a high RNA integrity.
The Kit method is the effective method in isolating RNA from inner scales of L. davidii var. unicolor. In inner scales of L. davidii var. unicolor, the absorbance spectra revealed that the A260/A230 ratios (ranged from 0.27 to 0.4) of RNA samples isolated by the modified TRIzol method were lower than that by the Kit (ranged from 1.96 to 2.06) and CTAB (ranged from 1.74 to 1.92) methods, but were higher than that by the TRIzol method (ranged from 0.19 to 0.22) ( Table 2). Additionally, only the A260/A280 ratios of RNA segregated by the TRIzol method were below 2.00 (ranged from 1.85 to 1.95), and the A260/A280 ratios of RNA gained by the other three methods were ranged from 2.10 to 2.28 (Table 2). These results suggested that the RNA samples isolated by the Kit and CTAB methods contained lower protein, organic solvents, and other contaminants compared to the TRIzol and modified TRIzol methods. Moreover, the Kit method could obtain a higher RNA  (Table 2). As for 1% agarose gel electrophoresis analysis, we failed to detect any RNA bands with the RNA samples obtained by the TRIzol method ( Fig. 2A). Interestingly, the RNA bands from the Kit and modified TRIzol methods were clear and intact, testifying that these RNA samples were not degraded (Fig. 2B,C). However, the separated RNA from the CTAB method was degraded obviously, on account of the lengthy procedure of this method (Fig. 2D). Thus, the Kit method is an optimized method to obtain high-quality total RNA from inner scales of L. davidii var. unicolor (Table 2; Fig. 2). In comparison with the TRIzol and CTAB methods, the modified TRIzol method could also extract high-quality total RNA from the inner scales.
High quantity and integrity RNA was obtained through the modified TRIzol method from the middle and external scales. For middle and external scales of L. davidii var. unicolor, the A260/A230 ratios of RNA samples ranged from 0.29 to 1.39, from 1.85 to 1.99, and from 1.55 to 1.89 for the modified TRIzol method, the Kit method and the CTAB method, respectively ( Table 3), indicating that the RNA samples extracted by the modified TRIzol and CTAB methods were contaminated by polyphenols and polysaccharides. There was no significant difference in A260/A280 ratios among these three methods, and the values were all higher than 2.0, demonstrating that all of the RNA samples were free of proteins and DNA contaminations. However, it is remarkable that when compared with the Kit and CTAB methods, the modified TRIzol method obtained the most enriching total RNA (ranged from 460.92 to 1581.90 ng μL −1 ) (Table 3).
Meanwhile, 1% agarose gel electrophoresis showed that the RNA bands were clear and complete from the modified TRIzol method and the Kit method (Figs. 3A,B, 4A,B). Nevertheless, the total RNA obtained by use Kit method (C) and CTAB method (D) were separated and analyzed in 1% agarose gels, respectively. Then the gels were visualized and exposed by Gel Imager System in different fields, and pictures were taken, respectively. The order of samples in each gel lane is as follows: Lane 1: DL2000 DNA marker; Lanes 2-4 are RNA isolated from root of Lilium; Lanes 5-7 are RNA isolated from stem of Lilium; Lanes 8-10 are RNA isolated from leaf of Lilium; Lane 11: Loading buffer solution without RNA to serve as a control. 28S and 18S represent the location of 28S and 18S rRNA bands. www.nature.com/scientificreports/ of the CTAB method was degraded seriously (Figs. 3C, 4C). Taken together, total RNA concentrations obtained from the middle and external scales by using the modified TRIzol method were higher than the other methods, and the RNA bands were clear and intact (Table 3; Figs. 3,4). What is noteworthy is that the RNA separated by all these methods from the basal part of Lilium scales showed higher purity and concentration than from other parts of the scales.
The modified TRIzol method is preferable for obtaining RNA with high quantity from the inner scales of other edible bulbs. In order to evaluate whether the modified TRIzol method is effective in extracting RNA from other kinds of edible bulbs, the TRIzol and modified TRIzol methods were employed simultaneously to gain RNA samples from the inner scales of Lilium lancifolium Thunb. and Lilium brownii var. viridulum Baker. As for samples of Lilium lancifolium Thunb., all the A260/A230 ratios were below 0.5. The  www.nature.com/scientificreports/ A260/A230 ratios of RNA samples from the TRIzol method were improved mildly compared with that from the modified TRIzol method ( Table 4). The A260/A280 ratios of the RNA samples extracted from the top-inner scales were similar for the TRIzol and modified TRIzol methods (2.11 and 2.12, respectively), whereas the A260/ A280 ratios of RNA samples from middle-inner scales and basal-inner scales for the modified TRIzol method (1.95 and 1.99, respectively) were significant higher than that for the TRIzol method (1.56 and 1.71, respectively). In addition, the RNA concentration of middle-inner scales from the modified TRIzol method (88.53 ng μL −1 ) was higher than that from the TRIzol method (53.11 ng μL −1 ), while the yields of RNA of top-inner scales and basal-inner scales from the modified TRIzol method were relatively low (Table 4). Similarly, the A260/A230 and A260/A280 ratios of RNA from inner scales of Lilium brownii var. viridulum Baker for the modified TRIzol method were relatively higher than that for the TRIzol method, and the RNA concentrations from different parts of the inner scales extracted by the modified TRIzol method (ranged from 45.17 to 87.79 ng μL −1 ) were significantly higher than that by the TRIzol method (ranged from 9.24 to 35.90 ng μL −1 ) (Table 4). Further, the RNA bands from these two edible bulbs were clear and complete gained from the modified TRIzol method in 1% agarose gel electrophoresis analysis (Fig. 5), while RNA bands extracted from the TRIzol method were weak or absent (Fig. 6). In all, the above results suggest that more sufficient total RNA can be obtained by applying the modified TRIzol method in edible bulbs of different Lilium species, though the contamination of polysaccharides, polyphenols and proteins were similar in RNA samples extracted from the two TRIzol methods.

Discussion
L. davidii var. unicolor has been regarded as both the vegetable and the herbal medicine for several centuries in China because its bulb could provide polysaccharides and phenolic compounds to treat bronchitis, pneumonia and provide nutritions 22,23 . Amounts of polyphenols and polysaccharides are accumulated during the bulb formation and development stages of Lilium. Hence, extracting high-quality RNA samples from Lilium bulb is a tough process as polysaccharides and polyphenols tend to co-precipitate with RNA 24,25 . The metabolite composition of plant tissues varies greatly, so different approaches are required to obtain high-quality RNA 16,26 . Several RNA extraction methods have been reported in seed or storage root of pistachio 14 , cassava 16 , Arabidopsis 19 , sunflower 27 , Jerusalem artichoke 24 and sugar beet 28 , including the TRIzol method. Over the past decade, TRIzol, Kit and CTAB methods were used to isolate total RNA from leaf, root and bulb of Lanzhou lily [19][20][21]29 . However, which method is more suitable of extracting total RNA from different tissues in this kind of Lilium is still needed to be explored. In this study, several methods were researched to obtain high-quality and integrated total RNA in an easy and low-cost condition from different tissues in Lilium, and the optimal RNA isolation method was selected and optimized. www.nature.com/scientificreports/ In the present study, the TRIzol method was introduced to isolate total RNA from root, stem, leaf and inner scales of Lanzhou lily, but resulted in low yield and divergent degradation (Figs. 1, 2). Meanwhile, we initially tried to extract RNA samples from different tissues of Lilium by applying the CTAB and Kit approaches, but the results remained unsatisfactory (Fig. 1). However, compared with the CTAB and Kit methods, the modified TRIzol method could extract high-quality RNA from different tissues of Lilium (Table 1). A previous study reported an improved TRIzol method to obtain total RNA from the bulb of Lanzhou lily, but it was failed to remove polysaccharides in Lilium 17 . Another version of modified TRIzol method could effectively eliminate the above contaminations by adding sodium acetate solution, DTT and β-mercaptoethanol reagents in the extracted procedure 19 , suggesting the potential role of TRIzol method in extracting RNA from Lanzhou lily. During the operation of the modified TRIzol method in this study, after the RNA was precipitated by isopropanol, changing the extraction environment to 4 °C, using the RNA purification adsorption column and washing the RNA pellet with ethanol repeatedly are critical procedures for obtaining high quality and purity RNA samples. Furthermore, large amount of TRIzol reagents should be used to effectively dissolve tissue samples and gain more supernatant, resulting in an increased quantity of total RNA. Among these testified methods, when using the modified TRIzol   www.nature.com/scientificreports/ method, the highest RNA concentration was obtained from root, stem, leaf (Table 1) and different parts of scales (Tables 2, 3) in Lilium, with the A260/A280 ratios ranging from 1.97 to 2.27. Although the A260/A230 ratios for different tissues were still low, the 28S and 18S rRNA bands could be clearly observed, confirming the integrity of the RNA samples isolated by the modified TRIzol method (Figs. 1, 2, 3, 4). These results were further certified by comparing the modified TRIzol method with the TRIzol method in other edible bulb samples (Lilium lancifolium Thunb. and Lilium brownii var. viridulum Baker) (Figs. 5, 6). The relatively lower RNA concentrations in these edible bulbs in comparison with Lanzhou lily may be caused by the long-term storage and transportation, while bulbs of Lanzhou lily were collected locally. In addition, for different scales of Lilium, it was easier to obtain high yield and quality total RNA from the basal part of the scales (Tables 2, 3). This might because that  viridulum Baker isolated using TRIzol method. The RNA samples extracted by TRIzol method were separated and analyzed in 1% agarose gels, respectively. Then the gels were visualized and exposed by Gel Imager System in different fields, and pictures were taken, respectively. www.nature.com/scientificreports/ the basal part have the higher transcriptional activity and less content of polysaccharides and other secondary metabolites than other parts of the scales. Previous studies have reported that CTAB method could extract high purity and yield of total RNA from different species which contained high content of polyphenols and polysaccharides 30,31 . Also, the CTAB method could obtain higher quality and quantity total RNA from leaf, root and bulb of Lanzhou lily 17,29 . In this study, while isolating RNA samples with the CTAB method from root, stem and leaf, one of the main obstacles is the existence of DNA contamination (Fig. 1). To further eliminate the interference of DNA from RNA samples, we added the RNA-free DNase I digestion step in the CTAB method for extracting RNA from different lily scales. In the scales of Lilium, as shown in Tables 2 and 3, the A260/A230 ratios of RNA samples isolated by the CTAB method were lower than that by the Kit method, but higher than that by the modified TRIzol method. When using the CTAB method, the A260/A280 ratios of the separated RNA samples were above 2.1 except for RNA samples of stem and leaf. The above results indicate that the CTAB method could effectively remove proteins, but couldn't avoid the contamination of polysaccharides and polyphenols in the isolated total RNA samples. Unexpectedly, the RNA bands in 1% agarose gel electrophoresis gained from the CTAB method were incomplete and diffused (Figs. 2, 3, 4), indicating that the RNA samples from the scales isolated by the CTAB method were degraded, though the contamination of DNA was obviously reduced. So we speculated that this might be due to the long time spent in the process of RNA extraction by the CTAB method, which led to the degradation of RNA. As reported before, the improved CTAB method is suitable for extracting high purity, intact and high yield RNA than the improved TRIzol method in Lilium 30 . However, compared with the modified TRIzol method, the CTAB method here obtained lower concentration and lower yield RNA in Lanzhou lily (Tables 1, 2, 3). The yield of RNA extracted by the CTAB method is reduced due to the high affinity of CTAB with nucleic acids and other biopolymers 32 . In addition, the process of the CTAB method is more complicated and more preparations are needed before the experiment compared with the modified TRIzol method, as the modified TRIzol method is easier to be conducted, and no solution except for 75% ethanol is needed to be prepared before. Thus, the modified TRIzol method may be more suitable than the CTAB method for acquiring total RNA samples from L. davidii var. unicolor.
Compared with the CTAB method, the Kit method resulted in higher yield and purity RNA from bulb scales that contain high levels of polysaccharides and polyphenols. Previous studies reported that Kit method could be used to isolate RNA from bulb of Lanzhou lily 20,21 . However, the Kit protocol is not suitable for extracting total RNA from root, stem and leaf of Lilium in the current study (Table 1). Meanwhile, the Kit protocol is the most expensive method among these methods, considering the extra amount of reagents used for extracting high quality and purity RNA from L. davidii var. unicolor. In our results, the modified TRIzol method could obtain more enriching and integrated RNA samples, though the RNA contains a contamination of polysaccharides and polyphenols. Together with the fact that the Kit method is overspent, and there is a complicated and interminable process within the CTAB method, we suggested that the modified TRIzol protocol is the efficient, simple and economical method to acquire high-quality and integrity total RNA from different tissues in Lilium.

Conclusion
TRIzol method, modified TRIzol method, CTAB method and Kit method were used to extract RNA from tissues in L. davidii var. unicolor, and the highest quality and yield total RNA were obtained from the modified TRIzol protocol. In addition, the intense and intact RNA bands in 1% agarose gel electrophoresis of the modified TRIzol method showed a distinct advantage among these methods. Furthermore, this method is an easy, efficient, and low-cost method for total RNA isolation from Lilium. In all, the modified TRIzol method is sufficient to gain eligible RNA in Lilium to support further molecular experiments.

Materials and methods
Plant materials. Lily plants 'L. davidii var. unicolor' were cultivated in the greenhouse (the average temperature was 18.5 °C and the average degree of humidity was 79.4%) of Gansu Agricultural University, Lanzhou, Gansu Province, China (36.10384 N, 103.7189 E). Firstly, fresh plants were collected and washed with deionized sterile water and dissected leaf, stem, root and bulb separately. Secondly, scales were removed carefully from mother bulb and sorted into three groups: external scales (1-3 layers), middle scales (4-6 layers) and inner scales (7-9 layers). Then different scales were sorted into three equal parts, named as top part, middle part and basal part (Fig. 7A). Thus, the different tissues from Lilium were named successively as top-inner scales, middle-inner scales, basal-inner scales, top-middle scales, middle-middle scales, basal-middle scales, top-external scales, middle-external scales and basal-external scales. Lily bulbs of Lilium lancifolium Thunb. were produced in Enshi, Hubei Province, China (110.09242 N, 30.2223 E) and harvested after the aerial part of the plants withered in October 2021. Lily bulbs of Lilium brownii var. viridulum Baker were produced in Longhui, Hunan Province, China (111.03249 N, 27.1140 E) and harvested after the aboveground part of the plants withered in August 2021. Different layers of the tested inner scales of these two edible bulbs were collected by the same method used in L. davidii var. unicolor (Fig. 7B,C). The above samples were immediately frozen in liquid nitrogen and then preserved at − 80 °C. All experiments conducted in this study, including the collection of plant materials, were in compliance with relevant institutional, national, and international guidelines and legislations.  2017), and the modified TRIzol method was done according to this TRIzol method with some modifications 33 . In order to obtain higher concentration of total RNA, RNA purification adsorption column were used after Steps 4. Our modified procedure is described as follows: (1) About 300 mg of each tissue samples was ground into a fine powder using liquid nitrogen and transferred into a 1.5 mL centrifuge tubes. Then 1 mL of TRIzol reagent was added, and mixed by a micropipettor. Incubated the samples for 10 min at 4 °C, and centrifuged at 12,000 g for 5 min at 4 °C. (2) The supernatant was transferred to a new 1.5 mL Eppendorf tube. 200 µL of cold chloroform was added and the mixture was hand-shaken and then incubated at room temperature for 5 min. Then it was centrifuged at 12,000 g for 15 min at 4 °C. (3) The supernatant (~ 700 μL) was transferred to a new 1.5 mL chilled Eppendorf tube and then an equal volume of chilled isopropanol was added and mixed by inverting the tube for 6-10 times. The final mixture was incubated at − 20 °C overnight to precipitate RNA. (4) The obtained RNA pellet from two repeats above was added to one RNA purification adsorption column (700 μL for each time) followed by a centrifugation of 15 min at 12,000 g at 4 °C to obtain a visible precipitate, and the supernatant was discarded. (5) 700 µL pre-cooled 75% ethanol were added and mixed by inverting the tube for 4-6 times, and centrifuged at 12,000 g for 1 min at 4 °C, then the supernatant were discarded. The ethanol washing step was repeated twice, then centrifuged at 12,000 g for 2 min at 4 °C and the redundant ethanol was discarded. (6) After centrifugation, the tubes were carefully transferred to the ice, and the washed RNA pellet was air-dried for 10 min in super clean bench. (7) 40 μL RNase free H 2 O was added onto the membrane of the RNA purification adsorption column to dissolve RNA. RNA samples were incubated at 4 °C for 3 min, and then centrifuged at 12,000 g for 2 min at 4 °C to elute RNA. To obtain high concentration of RNA, the first eluent was added back into the RNA purification adsorption column, incubated and centrifuged again as above.

Reagents and solutions.
Method 2: CTAB method. CTAB method was constructed according to White et al. (2008) with some modifications 10 , the RNase-free DNase I and RNA purification adsorption column were used in Steps 10 and 11, respectively. Our modified procedure is described as follows: (1) The extraction buffer was preheated in a 50 mL Eppendorf tube (with 3% β-mercaptoethanol added) and bathed at 65 °C for 30 min. (2) About 300 mg of each sample was grounded into a fine powder by using liquid nitrogen, then the sample was transferred to 1.5 mL chilled Eppendorf tube and 1.2 mL pre-heated extraction buffer was added. (3) The tube was vortexed immediately and then incubated at 65 °C in water bath for at least 30 min with vortexing once every 5 min. (4) The tube was centrifuged at 12,000 g for 10 min at 4 °C and the supernatant was transferred to a new 1.5 mL Eppendorf tube. (5) An equal volume (650-850 µL) of C: I (24:1) was added to the tube, with vortexing for 30 s and then centrifuged at 12,000 g for 15 min at 4 °C. (6) The aqueous phase was transferred to a new 1.5 mL Eppendorf tube without disruption of the white interphase. (7) Repeated C: I abstraction as in the previous steps (steps 5 and 6). (8) 1/3 volumes of 8 M pre-cooled LiCl was added to the supernatant in 1.5 mL Eppendorf tube and was mixed upside down, then the mixture was stored at 4 °C overnight for nucleic acid precipitation. (9) The obtained RNA pellet from two repeats above was combined and added to one RNA purification adsorption column (700 μL for each time) followed by a centrifugation of 15 min at 12, 000 g at 4 °C, then discarded the supernatant. (10) 5 µL 10 × DNase I Buffer, 4 µL Recombinant DNase I (TaKaRa, Tokyo, Japan, RNase free, 5 U µL −1 ) and 41 µL RNase free H 2 O were pipetted onto the membrane of the RNA purification adsorption column, followed by an incubation of 15 min at room temperature (25 °C) to eliminate possible genomic DNA contamination. (11) The RNA was washed, air-dried and eluted as described in steps 5-7 of the modified TRIzol protocol.  www.nature.com/scientificreports/ method was modified when using RNA purification adsorption column to collecting RNA. In order to ensure high quality total RNA, the same volume of the treatment mixture were collected to the same adsorption column as in method 1 and 2. Importantly, all of the operating steps were performed at 4 °C to prevent RNA degradation during the experiment process.
RNA assessment. The A230/260 and A260/280 ratios were measured by a Nano drop spectrophotometer to determine the purity of the extracted total RNA. The concentration of the RNA sample was detected simultaneously by the spectrophotometer to determine the RNA yield. RNA integrity was evaluated by the clarity of ribosomal RNA bands in 1% agarose gel electrophoresis 32 . The gels were stained with ethidium bromide (0.5 µg mL −1 ) and then visualized and documented by a Gel Imager System (GE, USA).

Data availability
All data generated or analyzed during this study are included in this article.