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Journal of Shellfish Research, Vol. 37, No. 1, 199–205, 2018.
MI-JIN CHOI,1 DO-YEON RHO,1 TAE HYUG JEONG,2 HAN KYU LIM2*
AND JONG-MYOUNG KIM1*
1
Department of Fishery Biology, PuKyong National University, YongSoRo 45, NamGu, Busan 48513,
Korea; 2Department of Marine and Fisheries Resources, Mokpo National University, YeongSanRo 1666,
Muan 58554, Korea
ABSTRACT RNA isolation from the hepatopancreas of adult abalone is challenging, as indicated by the variable results of
reverse transcription-polymerase chain reaction (RT-PCR) amplification. In this study, three RNA isolation methods were tested
to evaluate their usefulness in RNA isolation from various abalone tissues. Unlike the conventional method (method I), methods
II and III included additional centrifugation and lithium chloride precipitation steps, respectively. RNA quality assessment based
on the RT-PCR amplification of ribosomal protein L3 (RPL3) showed little difference in the quality of RNA isolated from muscle,
regardless of the isolation method used. While RNA isolated from the hepatopancreas using methods I and II resulted in lower
levels of RPL3 amplification, method III was found to be a more effective method of RNA extraction from the hepatopancreas, as
indicated by a 30–60-fold increase in the RPL3 level. Competitive inhibition experiments using mixtures of RNA prepared by each
method were performed to investigate which RT-PCR step was inhibited by materials present in the RNA preparation. An almost
complete absence of RPL3 amplification was observed in RT-PCR with a cDNA template prepared from a mixture of RNAs
containing a small amount of hepatopancreatic RNA isolated using method I. A drastic decrease in the incorporation of
a fluorescence-labeled nucleotide into cDNA was also evident in the reverse transcription containing hepatopancreatic RNA isolated
using method I. These results indicate a distinct inhibition of cDNA synthesis by materials present in the hepatopancreatic RNA
preparation isolated using method I, but not present when method III is used. A competitive inhibition experiment was performed
using mixtures of cDNA prepared from RNA isolated by each method to investigate whether the materials used in the preparation
inhibited the polymerase chain reaction (PCR) step. The level of RPL3 amplification was proportional to the amount of cDNA
prepared from hepatopancreatic RNA isolated using method III. The comparable degree of amplification regardless of the
amount of cDNA prepared from RNA isolated using method I, together with the low cDNA synthesis in the latter, indicated that
it had little, if any, inhibitory effect on the PCR step. This study reports an RNA isolation method that is applicable to the
hepatopancreas and other abalone tissues with similar efficacy.
KEY WORDS: Haliotis discus hannai, Pacific abalone, hepatopancreas, RNA isolation, RT-PCR
199
200 CHOI ET AL.
the presence of 2 mL RNase-free DNase I (Roche Diagnostics) Competitive Inhibition Test for cDNA Synthesis
at 37°C for 15 min to remove DNA traces. RNA purification
To determine whether coprecipitated substances inhibit the
was performed using a Hybrid-R RNA purification column
reverse transcription process, cDNA was synthesized with
(GeneAll) according to the manufacturerÕs instructions. The
different ratios of RNA prepared from muscle using method
quantity of purified RNA was assessed by agarose gel electro-
III and the hepatopancreas using method I or III. The former
phoresis and a Microvolume Nucleic Acid spectrophotometer
was included as a positive control without inhibitory materials.
(ASP-2680, v. 4.1; ACTGene, Inc., Piscataway, NJ).
On dilution of the RNAs to 200 ng/mL, cDNA synthesis was
Reverse Transcription and PCR carried out with 2 mg 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
The concentration of total RNA was adjusted to 200 ng/mL each tissue. The level of cDNA synthesized was analyzed by
for cDNA synthesis. First-strand cDNA was synthesized using direct measurement of cDNA or by PCR amplification of
an M-MLV cDNA Synthesis Kit (Enzynomics) with 2 mg total RPL3.
RNA resuspended in 19 mL DEPC-treated water and 1 mL For direct measurement of the cDNA product synthesized,
80 mM oligo dT18. The mixture was incubated at 70°C for 5 min reverse transcription was performed with the addition of
before chilling on ice for 5 min. The reaction mixture was then 0.2 mM Cy5-dCTP to the reaction mixture containing 2 mM
adjusted to 40 mL by adding 20 mL of reverse transcriptase (RT) dNTP and RNA, as described previously. cDNA products were
Mix containing 4 mL 103 M-MLV RT buffer, 1 mL M-MLV further incubated at 65°C for 15 min on addition of 2.5 mL of
RT (200 units/mL), 4 mL deoxynucleotide (dNTP) mixture 1 M NaOH to degrade RNA and purified using the AccuPrep
(2 mM each), 0.5 mL RNase inhibitor (40 units/mL), and 10.5 mL PCR Purification Kit according to the manufacturerÕs protocol
DEPC-treated water, and the mixture was incubated at 42°C for 1 h. (Bioneer). The amount of Cy5-dCTP incorporated into cDNA
The reaction was terminated by inactivation at 72°C for 10 min and was determined by measuring the fluorescence in the eluate with
then stored at –20°C until further use. a spectrofluorometer (FP8300; JASCO) at 649/670-nm excita-
Polymerase chain reaction was performed using primers tion and emission wavelengths, respectively. The concentration
specific to ribosomal protein L3 (RPL3F: 5#-TGT CAC CAT of single-stranded cDNA obtained from cDNA synthesis at
CCT TGA GGC AC-3#, and RPL3R: 5#-CAG GAA CAG a scale 2.5 times larger was also determined using the Qubit
GCT TCT CCA GG-3#). Polymerase chain reaction was ssDNA Assay Kit and Qubit 3.0 fluorometer (Invitrogen,
performed in a 20-mL volume reaction containing 2 mL cDNA Carlsbad, CA).
template and 1 mM each of RPL3F and RPL3R primers in HiQ-
PCR Mix (Genotech). Polymerase chain reaction conditions RESULTS
consisted of an initial denaturation step at 95°C for 3 min,
together with 25 cycles of denaturation at 95°C for 30 sec, RNA Isolation Methods from Abalone Tissues
annealing at 55°C for 30 sec, and extension at 72°C for 30 sec.
The final extension was carried out at 72°C for 5 min. Poly- The development of molecular tools that can be applied to
merase chain reaction was conducted on a T100 thermal cycler abalone is important for examining the mechanism associated
(Bio-Rad) and the product was visualized using 1.5% agarose with gene expression. A conventional RNA isolation method
gel electrophoresis followed by ethidium bromide staining. (method I in Fig. 1) based on guanidinium isothiocyanate–
Quantitative polymerase chain reaction was also performed phenol–chloroform has been used successfully for most tran-
on the Exicycler 96 from Bioneer to confirm quantitative scriptomic analyses in abalone (Choi et al. 2015). To test the
comparisons. Polymerase chain reaction was carried out in a quality of RNA isolated from various abalone tissues, RT-PCR
20-mL volume reaction containing 10 mL TOPreal qPCR 23 amplification was carried out with primers specific for RPL3.
PreMIX (Enzynomics), 4 mL cDNA templates, and 1 mM each Figure 2 shows the drastically decreased RPL3 level in the RT-
of RPL3F and RPL3R primers. Reaction steps consisted of PCR analysis of RNA isolated from the hepatopancreas
preincubation at 50°C for 4 min, initial denaturation at 95°C for compared with other tissues subjected to method I. This result
15 min, and 40 cycles of denaturation at 95°C for 10 sec, indicated that the conventional RNA isolation method was
annealing at 55°C for 15 sec, and extension at 72°C for 30 sec. successfully applied to most abalone tissues with similar RNA
yields and integrity, but its application to some abalone tissues,
Competitive Inhibition Test especially hepatopancreatic tissue, proved challenging, as dem-
onstrated by the variation in RT-PCR yields.
To examine whether substances that coprecipitated with To overcome the problems associated with tissue-specific
RNA inhibited PCR amplification, RT-PCR amplification of discrepancies resulting in a lower RT-PCR yield, other RNA
RPL3 was performed using mixtures of cDNA templates. Each isolation methods were tested for further optimization. In
cDNA template was prepared from the same amount (2 mg) of comparison with RNA isolation method I, method II included
total RNA extracted from the hepatopancreas using method I the centrifugation of lysates before chloroform extraction and
or III, under the conditions described previously, and then method III further included LiCl precipitation, as described in
diluted 5-fold with DEPC-treated distilled water. Competitive Figure 1. Although no differences in RNA quality or yield were
PCR was performed using 10 mL of mixtures containing noted among most tissues regardless of the isolation method
different ratios (10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10) of cDNA (data not shown), a clear difference in quality was observed in
prepared from hepatopancreatic RNA extracted by method III RNA isolated from hepatopancreatic tissue among isolation
to cDNA prepared from hepatopancreatic RNA isolated by methods I, II, and III, as indicated by the PCR amplification of
method I. Polymerase chain reaction was carried out as de- RPL3 (Fig. 3). This difference in the efficacy of RNA isolation
scribed previously. method I was more evident in fully grown abalone aged at least
202 CHOI ET AL.
2 mM dNTP in reverse transcription reactions, the amount of synthesis experiment revealing a correlation between cDNA
cDNA prepared from muscle RNA and hepatopancreatic RNA synthesized and the level of RPL3 amplification indicate its
isolated using method III was estimated to be approximately inhibitory effect on cDNA synthesis.
30 ng cDNA. Compared with the level of fluorescence in cDNA
prepared from muscle (10:0), set as 100%, the levels of cDNAs Inhibitory Effects on PCR
prepared from RNA mixtures containing increasing amounts
(7.5:2.5, 5:5, 2.5:7.5, and 0:10 ratios) of hepatopancreatic RNA To examine the effects on another RT-PCR step, PCR was
isolated using method III accounted for 98.77% ± 3.51%, carried out with cDNA prepared from the same amount of
99.66% ± 3.56%, 97.61% ± 2.79%, and 95.22% ± 2.83%, hepatopancreatic RNA isolated using methods III and I. A
respectively. The result showing a similar level of cDNA PCR series was performed using 10:0, 9:1, 7:3, 5:5, 3:7, 1:9,
products prepared in the presence of an increasing proportion and 0:10 ratios of cDNA prepared from hepatopancreatic
of hepatopancreatic RNA isolated using method III indicates RNA isolated using method III and cDNA prepared from
a weak inhibitory effect of the latter on reverse transcription. In RNA isolated using method I (Fig. 6, lanes 1–7). The level of
contrast, the level of Cy5-dCTP incorporated into cDNA was RPL3 amplification was compared with that in control
drastically reduced as the hepatopancreatic RNA isolated using experiments performed using the same amount of cDNA
method I was introduced (Fig. 5, columns 7–10). This was prepared from hepatopancreatic RNA isolated by method
shown by relative fluorescence intensities of 6.68% ± 2.82%, III but without cDNA prepared from RNA isolated by method I
2.92% ± 2.96%, 4.88% ± 4.03%, and 5.92% ± 0.57% in cDNAs (lanes 8–14). Figure 6 shows that the level of RPL3 amplification
prepared from RNA mixtures containing 7.5:2.5, 5:5, 2.5:7.5, was proportional to the amount of cDNA prepared from
and 0:10 ratios of muscle RNA, respectively, together with hepatopancreatic RNA isolated by method III regardless of the
hepatopancreatic RNA isolated using method I. A much presence of cDNA prepared from RNA isolated by method I
greater decrease in cDNA synthesis on the addition of hepato- (lanes 2–7 versus lanes 9–14). This may have resulted from a defect
pancreatic RNA prepared using method I compared with the in cDNA synthesis in the latter leading to the absence of cDNA
level of muscle RNA template provides supporting evidence for template, as shown in Figure 5. Specifically, a comparable degree
its inhibitory effect on cDNA synthesis. A similar degree of of RPL3 amplification was observed in the mixtures containing
inhibition on cDNA synthesis was also confirmed by an in- the same amount of cDNA mixture prepared from RNA isolated
dependent cDNA quantitation assay analyzing the amount of by method III, together with a larger volume of cDNA reaction
cDNA synthesized using the Qubit single-strand DNA assay mixture prepared from RNA isolated using method I (lane 6) or
system (data not shown). These results from the direct mea- with the same amount of water (lane 13). The absence of any
surement of cDNA synthesized and the competitive cDNA further decrease in the amplification of cDNA was also seen with
a 9-fold excess volume of cDNA-defective reaction mixture pre-
pared from RNA isolated using method I, indicating its weak, if
any, inhibitory effect on the PCR step.
DISCUSSION
2006, Liu et al. 2007, Baranski et al. 2008). Since the texture and reaction containing excess volumes of cDNA reaction mixtures
chemical characteristics of the organs and tissues of molluscs prepared from the hepatopancreatic RNA isolated using method
are quite different from those of mammals and fish, it is critical I as compared with the level of amplification in the reaction
to confirm whether the molecular methods used in mammals containing the same amount of the former (e.g., lane 13 versus
and fish are applicable to abalone. Polymerase chain reaction is lane 6 in Fig. 6) indicates little, if any, inhibitory effect of the latter
one of the most powerful molecular tools used for the rapid and mixture on the PCR step. It is likely that an inhibitory effect on
sensitive detection of genes, and RT-PCR is used to measure the PCR step can be ruled out, as there was little cDNA template
gene transcription levels. To use RT-PCR as a semiquantitative synthesis in the reaction prepared from hepatopancreatic RNA
comparison method, it is critical to prepare substantially pure isolated using method I. Overall, the materials present in the
RNA from various tissues by excluding substances that may hepatopancreatic RNA preparation isolated with method I, but
interfere with downstream applications. One of the most removed using method III, affected the RT-PCR quantitation via
common RNA isolation methods used for a variety of animal an inhibitory effect on the reverse transcription process.
tissues is based on an acid guanidinium isothiocyanate–phenol– Various modifications in RNA isolation protocols were
chloroform solution (Chomczynski & Sacchi 1987) which acts developed to optimize RNA isolation from organisms and
as a strong protein denaturant and RNase inhibitor. This has organs that contain varying amounts of metabolites that are
led to the production of commercial solutions such as TRIzol inhibitory to processes, including pure RNA isolation and PCR.
(Invitrogen) and TriRNA reagent (Favorgen Biotech, Ping- These included RNA isolation from wheat pistils (Manickavelu
Tung, Taiwan) that are widely used for RNA isolation in most et al. 2007) and Porphyra perforata (Hong et al. 1995) based on
animal tissues. Although the conventional RNA isolation difficulties in eliminating the viscous polysaccharide and second-
method appeared to work efficiently for most abalone tissues ary metabolites limiting RNA analysis using conventional
in a transcriptomic study of the growth of the Pacific abalone TRIzol reagent (Bugos et al. 1995, Li & Trick 2005, Kansal
Haliotis discus hannai (Choi et al. 2015) and Haliotis midae (van et al. 2008). Other modifications have included buffers containing
der Merwe et al. 2011), RNA isolation from some abalone cetyltrimethylammonium bromide combined with phenol (Apt
tissues proved challenging, as demonstrated by variation in the et al. 1995, White et al. 2008) and polyvinylpyrrolidone and
RT-PCR yields among tissues (Fig. 2). To overcome the cesium chloride density gradient ultracentrifugation for isolating
problems associated with tissue-specific discrepancies resulting nucleic acids from the giant kelp, Macrocystis pyrifera, brown
in a lower RT-PCR yield, three RNA isolation methods were alga Ectocarpus variabilis, and various plant species (MacKay &
tested and optimized in this study. A comparative analysis of Gallant 1991, Salzman et al. 1999, Gao et al. 2001). Lithium
RT-PCR using RNA isolated from hepatopancreatic tissue by chloride efficiently recovers pure RNA from various cellular
isolation methods I, II, and III indicated up to 64-fold in- components such as polysaccharides and inhibitors of cDNA
hibition of RT-PCR by materials in the hepatopancreatic RNA synthesis at a concentration of 2–2.5 M (Barlow et al. 1963,
preparations. Cathala et al. 1983, Sambrook & Russell 2001, Hong et al. 1995,
Lower levels of RPL3 amplified by RT-PCR may result from Manickavelu et al. 2007). Many organic compounds such as
the presence of coprecipitated materials that inhibit downstream polymeric phenol, polysaccharides, and proteins including col-
processes, cDNA synthesis, and PCR. Competitive reverse lagen act as PCR inhibitors (Rossen et al. 1992). Some oysters
transcription experiments conducted using mixtures of RNA and other bivalves contain PCR inhibitors such as glycogen and
isolated from hepatopancreas samples isolated by methods I polysaccharides (Atmar et al. 1993, 1995, Richards 1999), and
and III showed almost complete inhibition of RPL3 amplification secondary metabolites that coprecipitate with RNA (Dellacorte
in RT-PCRs using cDNA prepared with a small added amount of 1994, Kansal et al. 2008, Vanessa et al. 2008). For invertebrates
hepatopancreatic RNA isolated using method I. Direct evidence that feed on alginate-rich brown algae, polysaccharides and
of the inhibitory effect on cDNA synthesis was obtained from secondary metabolites are expected to accumulate in their
reverse transcription performed in the presence of a fluorescently organs, including the hepatopancreas and hindgut (Brunet
labeled nucleotide. This result showed abundant cDNA synthesis et al. 1994, McGaw & Curtis 2013, El-Maklizi et al. 2014). This
in the reaction prepared from hepatopancreatic RNA isolated may explain the variation in RPL3 levels amplified by RT-PCR
using method III, but negligible cDNA production in the reaction using RNA isolated from the abalone hepatopancreas, a digestive
prepared from hepatopancreatic RNA isolated using method I, organ. To overcome problems resulting from polysaccharide-
demonstrating the inhibitory effect of the latter on cDNA rich tissues in bivalves, RNA isolation procedures for removing
synthesis (Fig. 5). To investigate whether materials present in such inhibitory materials are needed. Among the three methods
the cDNA reaction mixture inhibited another RT-PCR step, tested to evaluate the efficiency of RNA isolation from two
competitive inhibition PCR was carried out with mixtures of abalone tissues in this study, the results indicate that method III
cDNA templates prepared from the same amount of hepato- which includes additional centrifugation and LiCl precipitation
pancreatic RNA isolated using methods I and III. The almost steps, was superior in removing substances that mainly inhibit
complete absence of cDNA production in the mixture prepared cDNA synthesis from hepatopancreatic RNA. Thus, method III
from the hepatopancreatic RNA isolated using method I explains is an effective RNA isolation method applicable to all tissues
why the level of amplification was proportional to the amount of from abalone with similar efficacy for the quantitative compar-
cDNA prepared from the hepatopancreatic RNA isolated using ison of transcripts.
method III and the identical reactions seen in Figure 6 when
supplementing the latter with either varying quantities of the ACKNOWLEDGMENTS
former products (lanes 1–7) or water (lanes 8–14). The absence of
any decrease in the amplification of cDNA prepared from We would like to express our thanks to Mr. Bok-Ki Choi
hepatopancreatic RNA isolated using method III, even in the for his technical help. This work was supported by the
RNA ISOLATION FROM ABALONE TISSUES 205
Golden Seed Project (213008-05-2 -SB710) funded by the Administration (RDA), and Korea Forest Service (KFS).
Ministry of Agriculture, Food and Rural Affairs (MAFRA), D.-Y. Rho was supported by LED-Grant funded by the
Ministry of Oceans and Fisheries (MOF), Rural Development MOF, Korea.
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