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AN EFFICIENT METHOD FOR PURIFYING HIGH-QUALITY RNA FROM THE HEPATOPANCREAS OF THE PACIFIC ABALONE HALIOTIS DISCUS HANNAI.

INTRODUCTION

Abalone is regarded as one of the most commercially important aquaculture species in Korea, but after the rapid increase in aquaculture production in the early part of the 21st century, abalone production has decreased in recent years. To improve the economic value of the abalone aquaculture industry, it is important to overcome the problems associated with a deteriorating aquaculture environment in overcrowded facilities and inbreeding depression in cultured stocks. Optimization of the culture conditions and nutritional factors has also been suggested as a strategy for improving productivity (Hahn 1989, Britz et al. 1994, Floreto et al. 1996, Lopez et al. 1998, Nelson et al. 2002). It is also important to understand the biology of abalone better, including the complex genetic basis of its growth. This knowledge could help shorten the culture time needed to grow abalone to a marketable size, a factor that affects the economics of the aquaculture industry. Although limited information is available for certain molecular biological tools used to examine the physiological aspects of abalone, various transcriptomic and proteomic approaches have recently been used to obtain information on genes and proteins affecting growth which might be useful for selection of abalone strains with a faster growth rate. These include next-generation sequencing technologies that have become popular in analyses of altered gene expression in Haliotis midae (van der Merwe et al. 2011) and the Pacific abalone Haliotis discus hannai (Choi et al. 2015), leading to the discovery of candidate genes associated with differential growth in abalone.

The expression of genes involved in regulatory processes can be influenced by environmental conditions. To examine the expression profile of specific genes at the transcript level using techniques such as RNA sequencing and reverse transcription-polymerase chain reaction (RT-PCR), it is critical to isolate pure intact RNAs from target tissues without contamination by materials that inhibit downstream processes, including cDNA synthesis and polymerase chain reaction (PCR) (To 2000, Li et al. 2010). Most transcriptomic analyses in abalone have been performed using RNA isolation based on acid guanidinium isothiocyanate-phenol-chloroform extraction (Chomczynski & Sacchi 1987). Although such conventional RNA isolation methods have been applied successfully in most animal tissues, the quality of RNA seems to vary depending on the levels of endogenous metabolites and inhibitory materials present in the target tissues or organisms. In a previous study involving tissue-specific expression analysis in Pacific abalone (Choi et al. 2015), lower yields from RT-PCR amplification of transcripts from specific tissues, in particular the hepatopancreas of fully mature abalone, were observed compared with the yields from other tissues. This might be due to the coprecipitation of inhibitory substances, together with the RNA, present in the hepatopancreas, an organ that plays important roles in food digestion as well as nutrient absorption and metabolite storage.

To overcome such tissue-specific discrepancies in RT-PCR yields and to allow for quantitative comparisons, a more efficient method of isolating high-quality RNA that is suitable for all abalone tissues is needed. Such methods should be optimized for the purification of intact RNA from abalone, regardless of the type of tissue or organism, with consistent efficacy. Various modifications of RNA isolation protocols have been developed that use cetyltrimethylammonium bromide, cesium chloride, or lithium chloride (LiCl), depending on the target organism and tissue (MacKay & Gallant 1991, Apt et al. 1995, Salzman et al. 1999, Gao et al. 2001, White et al. 2008). In the present study, three RNA isolation methods were tested to improve RNA quality by including additional centrifugation steps, chloroform extraction, and LiCl precipitation. The efficiency of these methods was assessed in hepatopancreatic and muscle tissues from Pacific abalone, and the resulting isolated RNA was tested by RT-PCR. Competitive inhibition experiments indicated that inclusion of the LiCl precipitation step efficiently removed substances inhibitory to cDNA synthesis (Barlow et al. 1963, Sambrook & Russell. 2001). In this study, a suitable method for RNA isolation from the hepatopancreas of mature abalone, as well as other abalone tissues, with similar efficacy has been reported.

MATERIALS AND METHODS

Materials

Specimens of the Pacific abalone Haliotis discus hannai were obtained from the commercial Namchun Market in Busan, Korea, and the Abalone Research Institute of Jeolla Namdo Marine-Fisheries & Development Institute (Wando, Korea). For tissue-specific analysis, Pacific abalones aged at least 2 y after fertilization were used. Various tissues were dissected from abalones, frozen in liquid nitrogen, and stored at -80[degrees]C. Guanidinium isothiocyanate-phenol-based TriRNA reagent (FavorPrep) was obtained from Favorgen Biotech Corporation (Ping-Tung, Taiwan). RNase-free recombinant DNase I was purchased from Roche Diagnostics (Indianapolis, IN), and Hybrid-R was obtained from GeneAll Corporation (Seoul, Korea) for RNA purification. Oligonucleotides and 5 x HiQ-PCR mix were obtained from Genotech (Daejeon, Korea) and M-MLV cDNA synthesis kits were purchased from Enzynomics (Daejeon, Korea). Quantitative polymerase chain reaction (qPCR) was performed using Exicycler 96 from Bioneer (Daejeon, Korea) and Cy5-dCTP (PA55021) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

RNA Isolation Methods

Three RNA isolation methods (Fig. 1) were tested to evaluate and compare their efficiency.

Method I

Method I is one of the most common methods of RNA extraction from animal tissues, using an acid guanidinium thiocyanate-phenol-chloroform solution. Up to 100 mg frozen tissue was homogenized using a tissue homogenizer in the presence of 1 mL TriRNA reagent. Chloroform (200 [micro]L) was added to the homogenate and the mixture was centrifuged at 15,000 g for 5 min at room temperature. The aqueous phase was collected into a new microcentrifuge tube and mixed with an equal volume of isopropanol followed by incubation at room temperature for 5 min. After centrifugation at 15,000 g for 5 min, the pellet was washed with 70% ethanol, dried, and resuspended in 100 [micro]L diethylpyrocarbonate (DEPC)-treated water.

Method II

Up to 100 mg frozen tissue was homogenized in 1 mL TriRNA reagent, as in method I, and the homogenate was centrifuged twice at 15,000 g for 5 min to remove cellular debris. Approximately 800 [micro]L of the supernatant was transferred into a new microcentrifuge tube, mixed with 160 [micro]L of chloroform, and centrifuged at 15,000 g for 5 min. The aqueous phase was transferred to a new tube, mixed with an equal volume of isopropanol, and incubated at room temperature for 5 min. RNA was precipitated by centrifugation at 15,000 g for 5 min and washed with 70% ethanol as described in method I.

Method III

Frozen tissue (~100 mg) was homogenized in 1 mL TriRNA reagent and centrifuged twice as described in method II. The homogenate supernatant (~800 [micro]L) was transferred to a new tube and mixed with 160 [micro]L chloroform, followed by centrifugation at 15,000 g for 5 min. The aqueous phase was mixed with an equal volume of isopropanol and incubated at room temperature for 5 min. After centrifugation at 15,000 g for 5 min, the pellet was washed with 70% ethanol. The dried pellet was resuspended in 400 [micro]L 5 M LiCl, followed by addition of 600 [micro]L DEPC-treated water. After incubation at -20[degrees]C for 1 h, RNA was precipitated by centrifugation at 15,000 g for 5 min. The pellet was washed with 70% ethanol, dried at room temperature, and then stored at -80[degrees]C after resuspension in 100 [micro]L DEPC-treated water.

DNase I Treatment and RNA Purification

RNA isolated by methods I-III was incubated in a total volume of 120 [micro]L in a solution containing 1 x DNase I buffer in the presence of 2 [micro]L RNase-free DNase I (Roche Diagnostics) at 37[degrees]C for 15 min to remove DNA traces. RNA purification was performed using a Hybrid-R RNA purification column (GeneAll) according to the manufacturer's instructions. The quantity of purified RNA was assessed by agarose gel electrophoresis and a Microvolume Nucleic Acid spectrophotometer (ASP-2680, v. 4.1; ACTGene, Inc., Piscataway, NJ).

Reverse Transcription and PCR

The concentration of total RNA was adjusted to 200 ng/[micro]L for cDNA synthesis. First-strand cDNA was synthesized using an M-MLV cDNA Synthesis Kit (Enzynomics) with 2 [micro]g total RNA resuspended in 19 [micro]L DEPC-treated water and 1 [micro]L 80 [micro]M oligo [dT.sub.18]. The mixture was incubated at 70[degrees]C for 5 min before chilling on ice for 5 min. The reaction mixture was then adjusted to 40 [micro]L by adding 20 [micro]L of reverse transcriptase (RT) Mix containing 4 [micro]L 10X M-MLV RT buffer, 1 [micro]L M-MLV RT (200 units/[micro]L), 4 [micro]L deoxynucleotide (dNTP) mixture (2 mM each), 0.5 [micro]L RNase inhibitor (40 units/[micro]L), and 10.5 [micro]L DEPC-treated water, and the mixture was incubated at 42[degrees]C for 1 h. The reaction was terminated by inactivation at 72[degrees]C for 10 min and then stored at -20[degrees]C until further use.

Polymerase chain reaction was performed using primers specific to ribosomal protein L3 (RPL3F: 5'-TGT CAC CAT CCT TGA GGC AC-3', and RPL3R: 5'-CAG GAA CAG GCT TCT CCA GG-3'). Polymerase chain reaction was performed in a 20-[micro]L volume reaction containing 2 [micro]L cDNA template and 1 [micro]M each of RPL3F and RPL3R primers in HiQ-PCR Mix (Genotech). Polymerase chain reaction conditions consisted of an initial denaturation step at 95[degrees]C for 3 min, together with 25 cycles of denaturation at 95[degrees]C for 30 sec, annealing at 55[degrees]C for 30 sec, and extension at 72[degrees]C for 30 sec. The final extension was carried out at 72[degrees]C for 5 min. Polymerase chain reaction was conducted on a T100 thermal cycler (Bio-Rad) and the product was visualized using 1.5% agarose gel electrophoresis followed by ethidium bromide staining. Quantitative polymerase chain reaction was also performed on the Exicycler 96 from Bioneer to confirm quantitative comparisons. Polymerase chain reaction was carried out in a 20-[micro]L volume reaction containing 10 [micro]L TOPreal qPCR 2x PreMIX (Enzynomics), 4 [micro]L cDNA templates, and 1 [micro]M each of RPL3F and RPL3R primers. Reaction steps consisted of preincubation at 50[degrees]C for 4 min, initial denaturation at 95[degrees]C for 15 min, and 40 cycles of denaturation at 95[degrees]C for 10 sec, annealing at 55[degrees]C for 15 sec, and extension at 72[degrees]C for 30 sec.

Competitive Inhibition Test

To examine whether substances that coprecipitated with RNA inhibited PCR amplification, RT-PCR amplification of RPL3 was performed using mixtures of cDNA templates. Each cDNA template was prepared from the same amount (2 [micro]g) of total RNA extracted from the hepatopancreas using method I or III, under the conditions described previously, and then diluted 5-fold with DEPC-treated distilled water. Competitive PCR was performed using 10 [micro]L of mixtures containing different ratios (10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10) of cDNA prepared from hepatopancreatic RNA extracted by method III to cDNA prepared from hepatopancreatic RNA isolated by method I. Polymerase chain reaction was carried out as described previously.

Competitive Inhibition Test for cDNA Synthesis

To determine whether coprecipitated substances inhibit the reverse transcription process, cDNA was synthesized with different ratios of RNA prepared from muscle using method III and the hepatopancreas using method I or III. The former was included as a positive control without inhibitory materials. On dilution of the RNAs to 200 ng/[micro]L, cDNA synthesis was carried out with 2 [micro]g of RNA from mixtures containing various (10:0, 7.5:2.5, 5:5,2.5:7.5, and 0:10) ratios of RNA isolated from each tissue. The level of cDNA synthesized was analyzed by direct measurement of cDNA or by PCR amplification of RPL3.

For direct measurement of the cDNA product synthesized, reverse transcription was performed with the addition of 0.2 mM Cy5-dCTP to the reaction mixture containing 2 mM dNTP and RNA, as described previously. cDNA products were further incubated at 65[degrees]C for 15 min on addition of 2.5 [micro]L of 1 M NaOH to degrade RNA and purified using the AccuPrep PCR Purification Kit according to the manufacturer's protocol (Bioneer). The amount of Cy5-dCTP incorporated into cDNA was determined by measuring the fluorescence in the eluate with a spectrofluorometer (FP8300; JASCO) at 649/670-nm excitation and emission wavelengths, respectively. The concentration of single-stranded cDNA obtained from cDNA synthesis at a scale 2.5 times larger was also determined using the Qubit ssDNA Assay Kit and Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA).

RESULTS

RNA Isolation Methods from Abalone Tissues

The development of molecular tools that can be applied to abalone is important for examining the mechanism associated with gene expression. A conventional RNA isolation method (method I in Fig. 1) based on guanidinium isothiocyanate-phenol-chloroform has been used successfully for most transcriptomic analyses in abalone (Choi et al. 2015). To test the quality of RNA isolated from various abalone tissues, RT-PCR amplification was carried out with primers specific for RPL3. Figure 2 shows the drastically decreased RPL3 level in the RT-PCR analysis of RNA isolated from the hepatopancreas compared with other tissues subjected to method I. This result indicated that the conventional RNA isolation method was successfully applied to most abalone tissues with similar RNA yields and integrity, but its application to some abalone tissues, especially hepatopancreatic tissue, proved challenging, as demonstrated by the variation in RT-PCR yields.

To overcome the problems associated with tissue-specific discrepancies resulting in a lower RT-PCR yield, other RNA isolation methods were tested for further optimization. In comparison with RNA isolation method 1, method II included the centrifugation of lysates before chloroform extraction and method III further included LiCl precipitation, as described in Figure 1. Although no differences in RNA quality or yield were noted among most tissues regardless of the isolation method (data not shown), a clear difference in quality was observed in RNA isolated from hepatopancreatic tissue among isolation methods I, II, and III, as indicated by the PCR amplification of RPL3 (Fig. 3). This difference in the efficacy of RNA isolation method I was more evident in fully grown abalone aged at least 3 y, in which the mature gonads are clearly visible; a lower level of RPL3 was amplified from hepatopancreatic RNA compared with RNA isolated from muscle, despite similar RNA yields and integrity (data not shown). Quantitative RT-PCR was also performed using RNA isolated from the hepatopancreas and muscle using methods I, II, and III for quantitative comparison. The threshold cycle (Ct) values for cDNA prepared from muscle RNA were approximately 20.84 [+ or -] 0.34 regardless of the isolation method used. By contrast, the Ct values for cDN A prepared from hepatopancreatic RNA isolated by methods I, II, and III were 25.03 [+ or -] 2.28, 24.87 [+ or -] 2.22, and 18.97 [+ or -] 1.03, respectively. This drastic difference in RNA quality demonstrated by Ct value differences of approximately 5 or 6 between methods I and II indicates up to 64-fold inhibition of RT-PCR by materials in the hepatopancreatic RNA preparation.

Inhibitory Effects on Reverse Transcription

Lower levels of RPL3 amplified by RT-PCR may result from the presence of coprecipitated materials that inhibit downstream processes, cDNA synthesis, and PCR. To examine their effects on cDNA synthesis, competitive reverse transcription experiments were conducted using mixtures of RNA isolated from the muscle using method III and RNA isolated from the hepatopancreas isolated by methods III and I. The RNA concentration was adjusted to 200 ng/[micro]L for a competitive inhibition experiment. For cDNA synthesis, RNA mixtures were prepared in a volume of 10 [micro]L at various ratios of 10:0, 7.5:2.5, 5:5, 2.5:7.5, and 0:10, containing muscle RNA without any inhibitory materials together with RNA isolated from the hepatopancreas by method III (lanes 1-5) and method I (lanes 6-10). Figure 4 shows a weak inhibitory effect on the amplification of RPL3 in reactions carried out with cDNA templates prepared from the RNA mixture containing various amounts of hepatopancreatic RNA isolated using method III, as shown by a slight decrease in the levels of RPL3. By contrast, almost complete inhibition of RPL3 amplification was observed in RT-PCRs using cDNA prepared with the addition of a small amount (even 1/4 of the RNA in the 10-[micro]L mixture; lane 7) of hepatopancreatic RNA isolated using method I (lanes 7-10). This result suggests that the materials present in the hepatopancreatic RNA prepared by method I inhibit RT-PCR.

To provide direct evidence for the inhibitory effect on cDNA synthesis, the same reverse transcription was performed in the presence of a fluorescence-labeled nucleotide, Cy5-dCTP. On purification of the cDNA, the amount of cDNA synthesized was directly determined by the fluorescence intensities of incorporated Cy5-dCTP-labeled nucleotides. A similar level of fluorescence intensity was detected in the cDNA prepared from muscle and hepatopancreas RNAs isolated using method III (Fig. 5, columns 1, 5, and 6). Based on the fluorescence intensity measured from an aliquot of a mixture containing 0.2 mM Cy5-dCTP and 2 mM dNTP in reverse transcription reactions, the amount of cDNA prepared from muscle RNA and hepatopancreatic RNA isolated using method III was estimated to be approximately 30 ng cDNA. Compared with the level of fluorescence in cDNA prepared from muscle (10:0), set as 100%, the levels of cDNAs prepared from RNA mixtures containing increasing amounts (7.5:2.5, 5:5, 2.5:7.5, and 0:10 ratios) of hepatopancreatic RNA isolated using method III accounted for 98.77% [+ or -] 3.51%, 99.66% [+ or -] 3.56%, 97.61% [+ or -] 2.79%, and 95.22% [+ or -] 2.83%, respectively. The result showing a similar level of cDNA products prepared in the presence of an increasing proportion of hepatopancreatic RNA isolated using method III indicates a weak inhibitory effect of the latter on reverse transcription. In contrast, the level of Cy5-dCTP incorporated into cDNA was drastically reduced as the hepatopancreatic RNA isolated using method I was introduced (Fig. 5, columns 7-10). This was shown by relative fluorescence intensities of 6.68% [+ or -] 2.82%, 2.92% [+ or -]2.96%, 4.88% [+ or -]4.03%, and 5.92% [+ or -]0.57% in cDNAs prepared from RNA mixtures containing 7.5:2.5, 5:5, 2.5:7.5, and 0:10 ratios of muscle RNA, respectively, together with hepatopancreatic RNA isolated using method I. A much greater decrease in cDNA synthesis on the addition of hepatopancreatic RNA prepared using method I compared with the level of muscle RNA template provides supporting evidence for its inhibitory effect on cDNA synthesis. A similar degree of inhibition on cDNA synthesis was also confirmed by an independent cDNA quantitation assay analyzing the amount of cDNA synthesized using the Qubit single-strand DNA assay system (data not shown). These results from the direct measurement of cDNA synthesized and the competitive cDNA synthesis experiment revealing a correlation between cDNA synthesized and the level of RPL3 amplification indicate its inhibitory effect on cDNA synthesis.

Inhibitory Effects on PCR

To examine the effects on another RT-PCR step, PCR was carried out with cDNA prepared from the same amount of hepatopancreatic RNA isolated using methods III and I. A PCR series was performed using 10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10 ratios of cDNA prepared from hepatopancreatic RNA isolated using method III and cDNA prepared from RNA isolated using method I (Fig. 6, lanes 1-7). The level of RPL3 amplification was compared with that in control experiments performed using the same amount of cDNA prepared from hepatopancreatic RNA isolated by method III but without cDNA prepared from RNA isolated by method I (lanes 8-14). Figure 6 shows that the level of RPL3 amplification was proportional to the amount of cDNA prepared from hepatopancreatic RNA isolated by method III regardless of the presence of cDNA prepared from RNA isolated by method I (lanes 2-7 versus lanes 9-14). This may have resulted from a defect in cDNA synthesis in the latter leading to the absence of cDNA template, as shown in Figure 5. Specifically, a comparable degree of RPL3 amplification was observed in the mixtures containing the same amount of cDNA mixture prepared from RNA isolated by method III, together with a larger volume of cDN A reaction mixture prepared from RNA isolated using method I (lane 6) or with the same amount of water (lane 13). The absence of any further decrease in the amplification of cDNA was also seen with a 9-fold excess volume of cDNA-defective reaction mixture prepared from RNA isolated using method I, indicating its weak, if any, inhibitory effect on the PCR step.

DISCUSSION

The development of molecular techniques that can be applied to abalone is important for understanding and characterizing its growth-related phenotypes (Chen et al. 2006, Lucas et al. 2006, Liu et al. 2007, Baranski et al. 2008). Since the texture and chemical characteristics of the organs and tissues of molluscs are quite different from those of mammals and fish, it is critical to confirm whether the molecular methods used in mammals and fish are applicable to abalone. Polymerase chain reaction is one of the most powerful molecular tools used for the rapid and sensitive detection of genes, and RT-PCR is used to measure gene transcription levels. To use RT-PCR as a semiquantitative comparison method, it is critical to prepare substantially pure RNA from various tissues by excluding substances that may interfere with downstream applications. One of the most common RNA isolation methods used for a variety of animal tissues is based on an acid guanidinium isothiocyanate-phenol-chloroform solution (Chomczynski & Sacchi 1987) which acts as a strong protein denaturant and RNase inhibitor. This has led to the production of commercial solutions such as TRIzol (Invitrogen) and TriRNA reagent (Favorgen Biotech, Ping-Tung, Taiwan) that are widely used for RNA isolation in most animal tissues. Although the conventional RNA isolation method appeared to work efficiently for most abalone tissues in a transcriptomic study of the growth of the Pacific abalone Haliotis discus hannai (Choi et al. 2015) and Haliotis midae (van der Merwe et al. 2011), RNA isolation from some abalone tissues proved challenging, as demonstrated by variation in the RT-PCR yields among tissues (Fig. 2). To overcome the problems associated with tissue-specific discrepancies resulting in a lower RT-PCR yield, three RNA isolation methods were tested and optimized in this study. A comparative analysis of RT-PCR using RNA isolated from hepatopancreatic tissue by isolation methods I, II, and III indicated up to 64-fold inhibition of RT-PCR by materials in the hepatopancreatic RNA preparations.

Lower levels of RPL3 amplified by RT-PCR may result from the presence of coprecipitated materials that inhibit downstream processes, cDNA synthesis, and PCR. Competitive reverse transcription experiments conducted using mixtures of RNA isolated from hepatopancreas samples isolated by methods I and III showed almost complete inhibition of RPL3 amplification in RT-PCRs using cDNA prepared with a small added amount of hepatopancreatic RNA isolated using method I. Direct evidence of the inhibitory effect on cDNA synthesis was obtained from reverse transcription performed in the presence of a fluorescently labeled nucleotide. This result showed abundant cDNA synthesis in the reaction prepared from hepatopancreatic RNA isolated using method III, but negligible cDNA production in the reaction prepared from hepatopancreatic RNA isolated using method I, demonstrating the inhibitory effect of the latter on cDNA synthesis (Fig. 5). To investigate whether materials present in the cDNA reaction mixture inhibited another RT-PCR step, competitive inhibition PCR was carried out with mixtures of cDNA templates prepared from the same amount of hepatopancreatic RNA isolated using methods I and III. The almost complete absence of cDNA production in the mixture prepared from the hepatopancreatic RNA isolated using method I explains why the level of amplification was proportional to the amount of cDNA prepared from the hepatopancreatic RNA isolated using method III and the identical reactions seen in Figure 6 when supplementing the latter with either varying quantities of the former products (lanes 1-7) or water (lanes 8-14). The absence of any decrease in the amplification of cDNA prepared from hepatopancreatic RNA isolated using method III, even in the reaction containing excess volumes of cDNA reaction mixtures prepared from the hepatopancreatic RNA isolated using method I as compared with the level of amplification in the reaction containing the same amount of the former (e.g., lane 13 versus lane 6 in Fig. 6) indicates little, if any, inhibitory effect of the latter mixture on the PCR step. It is likely that an inhibitory effect on the PCR step can be ruled out, as there was little cDNA template synthesis in the reaction prepared from hepatopancreatic RNA isolated using method I. Overall, the materials present in the hepatopancreatic RNA preparation isolated with method I, but removed using method III, affected the RT-PCR quantitation via an inhibitory effect on the reverse transcription process.

Various modifications in RNA isolation protocols were developed to optimize RNA isolation from organisms and organs that contain varying amounts of metabolites that are inhibitory to processes, including pure RNA isolation and PCR. These included RNA isolation from wheat pistils (Manickavelu et al. 2007) and Porphyra perforata (Hong et al. 1995) based on difficulties in eliminating the viscous polysaccharide and secondary metabolites limiting RNA analysis using conventional TRIzol reagent (Bugos et al. 1995, Li & Trick 2005, Kansal et al. 2008). Other modifications have included buffers containing cetyltrimethylammonium bromide combined with phenol (Apt et al. 1995, White et al. 2008) and polyvinylpyrrolidone and cesium chloride density gradient ultracentrifugation for isolating nucleic acids from the giant kelp, Macrocystis pyrifera, brown alga Ectocarpus variabilis, and various plant species (MacKay & Gallant 1991, Salzman et al. 1999, Gao et al. 2001). Lithium chloride efficiently recovers pure RNA from various cellular components such as polysaccharides and inhibitors of cDNA synthesis at a concentration of 2-2.5 M (Barlow et al. 1963, Cathala et al. 1983, Sambrook & Russell 2001, Hong et al. 1995, Manickavelu et al. 2007). Many organic compounds such as polymeric phenol, polysaccharides, and proteins including collagen act as PCR inhibitors (Rossen et al. 1992). Some oysters and other bivalves contain PCR inhibitors such as glycogen and polysaccharides (Atmar et al. 1993, 1995, Richards 1999), and secondary metabolites that coprecipitate with RNA (Dellacorte 1994, Kansal et al. 2008, Vanessa et al. 2008). For invertebrates that feed on alginate-rich brown algae, polysaccharides and secondary metabolites are expected to accumulate in their organs, including the hepatopancreas and hindgut (Brunet et al. 1994, McGaw & Curtis 2013, El-Maklizi et al. 2014). This may explain the variation in RPL3 levels amplified by RT-PCR using RNA isolated from the abalone hepatopancreas, a digestive organ. To overcome problems resulting from polysaccharide-rich tissues in bivalves, RNA isolation procedures for removing such inhibitory materials are needed. Among the three methods tested to evaluate the efficiency of RNA isolation from two abalone tissues in this study, the results indicate that method III which includes additional centrifugation and LiCl precipitation steps, was superior in removing substances that mainly inhibit cDN A synthesis from hepatopancreatic RNA. Thus, method III is an effective RNA isolation method applicable to all tissues from abalone with similar efficacy for the quantitative comparison of transcripts.

ACKNOWLEDGMENTS

We would like to express our thanks to Mr. Bok-Ki Choi for his technical help. This work was supported by the Golden Seed Project (213008-05-2 -SB710) funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA), and Korea Forest Service (KFS). D.-Y. Rho was supported by LED-Grant funded by the MOF, Korea.

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MI-JIN CHOI, (1) DO-YEON RHO, (1) TAE HYUG JEONG, (2) HAN KYU LIM (2*) AND JONG-MYOUNG KIM (1*)

(1) Department of Fishery Biology, PuKyong National University, YongSoRo 45, NamGu, Busan 48513, Korea; (2) Department of Marine and Fisheries Resources, Mokpo National University, YeongSanRo 1666, Muan 58554, Korea

(*) Corresponding authors. E-mails: limhk@mokpo.ac.kr and jongkim@pknu.ac.kr

DOI: 10.2983/035.037.0118
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Author:Choi, Mi-Jin; Rho, Do-Yeon; Jeong, Tae Hyug; Lim, Han Kyu; Kim, Jong-Myoung
Publication:Journal of Shellfish Research
Article Type:Report
Date:Apr 1, 2018
Words:5686
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