Evaluation of Six Methods for Extraction of Total RNA from Loquat

Trizol extraction for polysaccharide-rich plant tissue was unsuitable for isolating total RNA from loquat fruit. CTAB-LiCl extraction was improved with pretreatment using washing buffer with 80% ethanol and 70% acetone. The RNA isolated by this protocol from different loquat fruit tissue at various development periods, was high in purity (A260/A280 ratio ranged from 1.81 to 1.99 and A260/A230 ratio was over 2.0), and high in yield.


Introduction
Loquat (Eriobotrya japonica Lindl, Rosaceae, subfamily Maloideae), is indigenous to China, but is now cultivated worldwide in suitable climates.Loquat contains high level of polysaccharides, proteins, and secondary metabolites such as polyphenols (Liu et al., 2005) that cause degradation and low yield of functional mRNA (Wang et al., 2000).In this study, six methods for total RNA extraction of loquat fruit were compared and evaluated.

Materials
Fruit of loquat (Eriobotrya japonica Lindl.cv.'Zaozhong' No.6) were selected at different maturities from the germplasm collection of the College of Horticulture in South China Agricultural University, Guangzhou.The samples, including loquat peel and pulp, were frozen immediately by liquid nitrogen and stored at -70°C.Plastic ware were immersed in 0.1% (v/v) diethyl pyrocarbonate (DEPC) treated water at 37°C overnight and then autoclaved 1 h at 12°C.Glassware and the mortar were baked for 6 h at 20°C.The samples were ground into fine power in liquid nitrogen with a pre-cooled mortar.

Method 1
The protocol developed was based on the previously improved trizol extraction of Lin and Chen (2008).Frozen sample powder (0.2 g) was transferred to a 2 ml centrifuge tube with 1 ml RNA iso-mate for Plant Tissue (TaKaRa, Japan), mixed well, and then stored at room temperature for 5 min.Supernatant was collected after centrifuging at 12,000 g for 5min at 4°C to which an equal volume of Trizol and 1/10 volume of anhydrous ethanol was added, mixed well, and then centrifuged for 10 min at 12,000 g at 4°C.RNA crude product was extracted with 0.2 ml chloroform, vigorously vortexed and centrifuged at 12,000 g for 5 min at 4°C after incubated at room temperature for 5 min.The supernatant was transferred to a new tube and add an equal volume anhydrous ethanol was added.mixed well and centrifuged at 1,500 g for 10 min at 4°C after remaining at room temperature for 10min.The supernatant was discarded and 200 μl 4M LiCl added to suspended precipitate, placed in -20°C for 1 h.After centrifugation at 12,000 g for 20 min at 4°C, the RNA pellet was washed twice with 75% (v/v) alcohol, air-dried, then dissolved in 20 μl RNase-free water and stored at -70°C.
Method 2 RNAiso for polysaccharide-rich plant tissue (TaKaRa, Japan) was used as RNA extraction buffer.The procedure was performed according to the manufacturer's direction except that the RNA extraction buffer contained 2% (w/v) β-mercaptoethanol.The RNA pellet was dissolved in 20 μl RNase-free water and stored at -70°C.

Method 3
The protocol was modified based on the CTAB-LiCl extraction method of Song et al. (2011).Frozen sample powder (0.2 g) was transferred to a 2 ml centrifuge tube containing 1 ml CTAB extraction buffer [3% (w/v) CTAB, 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mm EDTA (pH 8.0), 2% (w/v) PVPP, 2% (w/v) β-mercaptoethanol and 80 μg•ml -1 proteinase K] mixed well and incubated at 65°C for 30 min with occasional shaking.The supernatant was collected by centrifuging at 8,000 g for 15 min at room temperature and an equal volume of chloroform-isoamyl alcohol (24:1, v/v) was added, mixed well and centrifuged at 12,000 g for 15 min at 4°C.The supernatant was collect-etone，2%(w/v)β-mercaptoethanol ], mix well and centrifuge at 8,000 g for 10 min at room temperature.Discard the supernatant, and then add 1 ml washing buffer into the precipitate and centrifuge at 8,000 g for 10 min again.After that, let the precipitate lysis in CTAB extraction buffer.The additional steps follow Method 3 as described.

RNA quantity and quality analysis
The integrity of total RNA was examined by electrophoresis on 1% formaldehyde denaturing agarose gels; sample loading volume was 3 μl.The purity and yield of total RNA isolated was assessed by measuring optical density (OD value) at 230, 260 and 280 nm using a spectrophotometer (BIO-RAD, Germany).

Results
Peel and pulp of young and ripe fruit as materials were evaluated with six methods of total RNA extraction.The yield and purity of total RNA in loquat from different stages were different based on the method used (Fig. 1 and Tab. 1).
With method 1, the total RNA of the young fruits had A 260 /A 230 ratios lower than 2.0, suggesting that RNA samples were little contaminated by polysaccharides, proteins, DNA, phenol or salts.With ripe fruit, total RNA was difficult to detected by gel electrophoresis (Fig. 1, A) indicating method 1 failed to isolate total RNA.
Method 2 isolated total RNA and yields, especially for the pulp of ripe fruits, was the highest (21.22 μg/g FW) of the six methods.However, the A 260 /A 230 ratio (0.87-1.45) was low and the electrophoresis bands were indistinct (Fig. 1, B).
Method 3 proved to be a practical method for RNA extraction.The RNA samples can be used for further study because their A 260 /A 280 and A 260 /A 230 ratios were 1.87-2.05and 2.32-3.62.Furthermore, distinct bands of 28S, 18S ri-ed again and 1/3 volume of 10 M LiCl was added, mixed well and incubated on ice overnight.After centrifugation at 12,000 g for 30 min at 4°C, the RNA precipitate was washed twice with 200 μl 4M LiCl.The precipitate was collected by centrifugation at 12,000 g for 30 min at 4°C and suspended with 200 μl of 10 mM Tris-HCl (pH 7.5), to which was added 1/10 volume of 3 mol•L -1 potassium acetate (pH 5.5) and kept on ice for 30 min, then separated by centrifuging at 12,000 g for 15 min at 4°C.The collected supernatant with the addition of 2.5 volume of cold anhydrous ethanol was stored for 2 to 3 h at -70°C.After centrifugation for 20 min at 12,000 g and 4°C, the RNA pellet was collected, washed twice with 75% (v/v) ethanol, air-dried, then dissolved in 20 μl RNase-free water and stored at -70°C.

Method 4
Frozen sample powder (0.2 g) was transferred into a 2 ml centrifuge tube, and 1 ml washing buffer [100 mMTris-HCl (pH 8.0), 0.35 M Sorbitol, 5 mM EDTA, 100 g•L -1 PEG6000] was added, mixed well and centrifuged at 8,000 g for 10 min at room temperature.The supernatant was discarded and, 1 ml 3% CTAB extraction buffer was added.The additional steps follow Method 3 as described.

Method 5
Frozen sample powder (0.2 g) were transferred to a 2 ml centrifuge tube, and before lysis in CTAB extraction buffer, 1 ml washing buffer [80% anhydrous ethanol，2% (w/v)β-mercaptoethanol]was added, mixed well and centrifuged at 8,000 g for 10 min at room temperature.Discard the supernatant, and add 1 ml washing buffer into the precipitate and centrifuge at 8000 g for 10 min again.The additional steps follow Method 3 as described.
In order to obtain high yield and high quality of total RNA, the sample powder was pre-processed with three different washing buffers before lysis in the CTAB extraction buffer.Methods 4, 5, and 6 were developed based on this concept.Compared with method 1 and method 2, total RNA isolated by using method 4, 5, and 6, were superior with high purity for loquat fruit.Their A 260 /A 280 ratios ranged from 1.81 to 2.08 and A 260 /A 230 ratios ranged from 2.29 to 3.68.Distinct bands of 28S and 18S ribosomal RNA were also observed in electrophoresis picture (Fig. 1, D, E, F).These results indicated that high quality total RNA could be isolated from peel or purl by pretreatment although yields different.

Discussion
Although there are many methods for RNA isolation, there is no universal one for all plant species or organs.Even identical tissues at different development stages may require a special protocol of RNA isolation due to the variability of chemical compositions and content (Sharma et al., 2003;Wang et al., 2007).Loquat is a recalcitrant woody fruit tree; its peel and pulp during differentiation contain more abundant and intricate polysaccharides, proteins, and secondary metabolites such as polyphenols than those of herbaceous plants (Liu et al., 2005), causing great difficulty to isolate total RNA with high quality and high yield.
The traditional Trizol kit which is the most common RNA isolation method is easy to operate and saves time and labor.Method 1 is a modification based on this kit and proved to be effective for RNA isolation from papaya and tomato fruits (He et al., 2010;Lin et al., 2008); however, this improved method failed to isolate total RNA from loquat fruit.The RNAiso kit is used for polysaccharide-rich plant tissue (TaKaRa, Japan) rich in polysaccharides and polyphenols can isolate RNA from loquat fruit.However, the low A 260 /A 230 ratio (0.87-1.45) and indistinct bands in electrophoresis picture (Fig. 1, B) indicated that RNA isolated by this method may contain many chemical substances, such as DNA, phenols, polysaccharides and salt, which could lead to negative effects.Song et al. (2011) reported that improved CTAB-LiCl extraction was a practicable protocol for loquat buds at different stages.Our study indicated that improved CTAB-LiCl extraction is satisfactory for loquat fruit.The only disadvantage is the low yield (6.46 μg/g FW) obtained from ripe fruit pulp.Previous studies suggested that high quality and high yield total RNA could be isolated from apricot (Li et al., 2010), plum (Xu et al., 2010)，litchi (Zhang et al, 2004), mango (Xiao et al., 2003;Zhang et al., 2009) and sisal (Zhang et al., 2010) when the sample powder was pre-processed by washing buffer before lysis in CTAB extraction buffer.Our study also confirmed that pretreatment with washing buffer, especially with 80% ethanol and 70% acetone, can effectively improve the quality of isolated RNA.We acknowledge that isolated RNA yields show slight differences based on method.For example, when using the 80% ethanol as the washing buffer, we obtained the highest yield of RNA (40.62 μg/g FW) from young fruit pulp using the 70% acetone, while highest yields were obtained from peels of both young (57.68 μg/g FW) and ripe fruits (40.86 μg/g FW).The experimental results demonstrated that total RNA isolated by method 5 and 6 were quite high in purity, proved to be suitable for fruit, and satisfactory for molecular downstream applications.
Tab. 1. Yield and absorbance ratio of total RNA extracted from loquat fruit with different methods