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An Efficient Method for Purifying High-Quality RNA from the Hepatopancreas of

the Pacific Abalone Haliotis discus hannai


Author(s): Mi-Jin Choi, Do-Yeon Rho, Tae Hyug Jeong, Han Kyu Lim and Jong-Myoung Kim
Source: Journal of Shellfish Research, 37(1):199-205.
Published By: National Shellfisheries Association
URL: http://www.bioone.org/doi/full/10.2983/035.037.0118

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Journal of Shellfish Research, Vol. 37, No. 1, 199–205, 2018.

AN EFFICIENT METHOD FOR PURIFYING HIGH-QUALITY RNA FROM THE


HEPATOPANCREAS OF THE PACIFIC ABALONE HALIOTIS DISCUS HANNAI

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

INTRODUCTION sequencing technologies that have become popular in analyses


of altered gene expression in Haliotis midae (van der Merwe
Abalone is regarded as one of the most commercially
et al. 2011) and the Pacific abalone Haliotis discus hannai (Choi
important aquaculture species in Korea, but after the rapid
et al. 2015), leading to the discovery of candidate genes
increase in aquaculture production in the early part of the 21st
associated with differential growth in abalone.
century, abalone production has decreased in recent years. To
The expression of genes involved in regulatory processes can
improve the economic value of the abalone aquaculture in-
be influenced by environmental conditions. To examine the
dustry, it is important to overcome the problems associated
expression profile of specific genes at the transcript level using
with a deteriorating aquaculture environment in overcrowded
techniques such as RNA sequencing and reverse transcription-
facilities and inbreeding depression in cultured stocks. Optimi-
polymerase chain reaction (RT-PCR), it is critical to isolate
zation of the culture conditions and nutritional factors has also
pure intact RNAs from target tissues without contamination by
been suggested as a strategy for improving productivity (Hahn
materials that inhibit downstream processes, including cDNA
1989, Britz et al. 1994, Floreto et al. 1996, Lopez et al. 1998,
synthesis and polymerase chain reaction (PCR) (To 2000, Li
Nelson et al. 2002). It is also important to understand the
et al. 2010). Most transcriptomic analyses in abalone have been
biology of abalone better, including the complex genetic basis of
performed using RNA isolation based on acid guanidinium
its growth. This knowledge could help shorten the culture time
isothiocyanate–phenol–chloroform extraction (Chomczynski &
needed to grow abalone to a marketable size, a factor that
Sacchi 1987). Although such conventional RNA isolation
affects the economics of the aquaculture industry. Although
methods have been applied successfully in most animal tissues,
limited information is available for certain molecular biological
the quality of RNA seems to vary depending on the levels of
tools used to examine the physiological aspects of abalone,
endogenous metabolites and inhibitory materials present in the
various transcriptomic and proteomic approaches have recently
target tissues or organisms. In a previous study involving tissue-
been used to obtain information on genes and proteins affecting
specific expression analysis in Pacific abalone (Choi et al. 2015),
growth which might be useful for selection of abalone strains
lower yields from RT-PCR amplification of transcripts from
with a faster growth rate. These include next-generation
specific tissues, in particular the hepatopancreas of fully mature
*Corresponding authors. E-mails: limhk@mokpo.ac.kr and jongkim@pknu. abalone, were observed compared with the yields from other
ac.kr tissues. This might be due to the coprecipitation of inhibitory
DOI: 10.2983/035.037.0118 substances, together with the RNA, present in the hepatopancreas,

199
200 CHOI ET AL.

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 bro-
mide, 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 centri-
fugation steps, chloroform extraction, and LiCl precipitation.
Figure 1. Diagram showing the principles of the three RNA isolation
The efficiency of these methods was assessed in hepatopancre-
methods evaluated in this study. Basic procedures used in the conventional
atic and muscle tissues from Pacific abalone, and the resulting
RNA isolation procedure (method I), together with additional centrifuga-
isolated RNA was tested by RT-PCR. Competitive inhibition tion steps (methods II) and LiCl precipitation (method III) are indicated.
experiments indicated that inclusion of the LiCl precipitation
step efficiently removed substances inhibitory to cDNA syn-
thesis (Barlow et al. 1963, Sambrook & Russell. 2001). In this 15,000 g for 5 min at room temperature. The aqueous phase was
study, a suitable method for RNA isolation from the hepato- collected into a new microcentrifuge tube and mixed with an
pancreas of mature abalone, as well as other abalone tissues, equal volume of isopropanol followed by incubation at room
with similar efficacy has been reported. temperature for 5 min. After centrifugation at 15,000 g for
5 min, the pellet was washed with 70% ethanol, dried, and
MATERIALS AND METHODS resuspended in 100 mL diethylpyrocarbonate (DEPC)-treated
water.
Materials
Method II
Specimens of the Pacific abalone Haliotis discus hannai were
Up to 100 mg frozen tissue was homogenized in 1 mL
obtained from the commercial Namchun Market in Busan,
TriRNA reagent, as in method I, and the homogenate was
Korea, and the Abalone Research Institute of JeollaNamdo
centrifuged twice at 15,000 g for 5 min to remove cellular debris.
Marine-Fisheries & Development Institute (Wando, Korea).
Approximately 800 mL of the supernatant was transferred into
For tissue-specific analysis, Pacific abalones aged at least 2 y
a new microcentrifuge tube, mixed with 160 mL of chloroform,
after fertilization were used. Various tissues were dissected from
and centrifuged at 15,000 g for 5 min. The aqueous phase was
abalones, frozen in liquid nitrogen, and stored at –80°C.
transferred to a new tube, mixed with an equal volume of
Guanidinium isothiocyanate–phenol-based TriRNA reagent
isopropanol, and incubated at room temperature for 5 min.
(FavorPrep) was obtained from Favorgen Biotech Corporation
RNA was precipitated by centrifugation at 15,000 g for 5 min
(Ping-Tung, Taiwan). RNase-free recombinant DNase I was
and washed with 70% ethanol as described in method I.
purchased from Roche Diagnostics (Indianapolis, IN), and
Hybrid-R was obtained from GeneAll Corporation (Seoul, Method III
Korea) for RNA purification. Oligonucleotides and 53 HiQ-
Frozen tissue (;100 mg) was homogenized in 1 mL TriRNA
PCR mix were obtained from Genotech (Daejeon, Korea) and
reagent and centrifuged twice as described in method II. The
M-MLV cDNA synthesis kits were purchased from Enzynom-
homogenate supernatant (;800 mL) was transferred to a new
ics (Daejeon, Korea). Quantitative polymerase chain reaction
tube and mixed with 160 mL chloroform, followed by centrifu-
(qPCR) was performed using Exicycler 96 from Bioneer
gation at 15,000 g for 5 min. The aqueous phase was mixed with
(Daejeon, Korea) and Cy5-dCTP (PA55021) was obtained
an equal volume of isopropanol and incubated at room
from Amersham Pharmacia Biotech (Piscataway, NJ).
temperature for 5 min. After centrifugation at 15,000 g for
RNA Isolation Methods 5 min, the pellet was washed with 70% ethanol. The dried pellet
was resuspended in 400 mL 5 M LiCl, followed by addition of
Three RNA isolation methods (Fig. 1) were tested to
600 mL DEPC-treated water. After incubation at –20°C for 1 h,
evaluate and compare their efficiency.
RNA was precipitated by centrifugation at 15,000 g for 5 min.
Method I The pellet was washed with 70% ethanol, dried at room
temperature, and then stored at –80°C after resuspension in
Method I is one of the most common methods of RNA 100 mL DEPC-treated water.
extraction from animal tissues, using an acid guanidinium
thiocyanate–phenol–chloroform solution. Up to 100 mg frozen DNase I Treatment and RNA Purification
tissue was homogenized using a tissue homogenizer in the
presence of 1 mL TriRNA reagent. Chloroform (200 mL) was RNA isolated by methods I–III was incubated in a total
added to the homogenate and the mixture was centrifuged at volume of 120 mL in a solution containing 13 DNase I buffer in
RNA ISOLATION FROM ABALONE TISSUES 201

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.

Inhibitory Effects on Reverse Transcription

Lower levels of RPL3 amplified by RT-PCR may result from


the presence of coprecipitated materials that inhibit down-
stream 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/mL for a competitive
inhibition experiment. For cDNA synthesis, RNA mixtures
were prepared in a volume of 10 mL 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
Figure 2. Tissue-specific differences in the quality of RNA isolated using
hepatopancreas by method III (lanes 1–5) and method I (lanes
the conventional method (method I). Reverse transcription-polymerase 6–10). Figure 4 shows a weak inhibitory effect on the amplifi-
chain reaction (RT-PCR) analysis was carried out using primers specific cation of RPL3 in reactions carried out with cDNA templates
to RPL3 and cDNA synthesized from equal amounts of RNA isolated prepared from the RNA mixture containing various amounts of
from tissues, including the ganglion (Gn), tentacle (Tn), gill (Gi), heart hepatopancreatic RNA isolated using method III, as shown by
(Ht), hepatopancreas (Hp), digestive tract (Dg), gonad (Gn), mantle a slight decrease in the levels of RPL3. By contrast, almost
(Mn), and muscle (Ms) of the Pacific abalone Haliotis discus hannai. The complete inhibition of RPL3 amplification was observed in RT-
sizes (kb) of the molecular weight markers (M) are indicated. PCRs using cDNA prepared with the addition of a small
amount (even 1/4 of the RNA in the 10-mL mixture; lane 7) of
3 y, in which the mature gonads are clearly visible; a lower level hepatopancreatic RNA isolated using method I (lanes 7–10).
of RPL3 was amplified from hepatopancreatic RNA compared This result suggests that the materials present in the hepato-
with RNA isolated from muscle, despite similar RNA yields pancreatic RNA prepared by method I inhibit RT-PCR.
and integrity (data not shown). Quantitative RT-PCR was also To provide direct evidence for the inhibitory effect on cDNA
performed using RNA isolated from the hepatopancreas and synthesis, the same reverse transcription was performed in the
muscle using methods I, II, and III for quantitative comparison. presence of a fluorescence-labeled nucleotide, Cy5-dCTP. On
The threshold cycle (Ct) values for cDNA prepared from purification of the cDNA, the amount of cDNA synthesized
muscle RNA were approximately 20.84 ± 0.34 regardless of was directly determined by the fluorescence intensities of in-
the isolation method used. By contrast, the Ct values for cDNA corporated Cy5-dCTP–labeled nucleotides. A similar level of
prepared from hepatopancreatic RNA isolated by methods I, fluorescence intensity was detected in the cDNA prepared from
II, and III were 25.03 ± 2.28, 24.87 ± 2.22, and 18.97 ± 1.03, muscle and hepatopancreas RNAs isolated using method III (Fig. 5,
respectively. This drastic difference in RNA quality demon- columns 1, 5, and 6). Based on the fluorescence intensity measured
strated by Ct value differences of approximately 5 or 6 between from an aliquot of a mixture containing 0.2 mM Cy5-dCTP and
methods I and II indicates up to 64-fold inhibition of RT-PCR
by materials in the hepatopancreatic RNA preparation.

Figure 4. Reverse transcription-polymerase chain reaction analysis per-


formed using cDNA templates prepared from mixtures of RNA isolated
from muscle and hepatopancreas of the Pacific abalone Haliotis discus
hannai. RNA was isolated from the muscle using method III and from the
Figure 3. Reverse transcription-polymerase chain reaction analysis of hepatopancreas using methods III and I. Reverse transcription was
RPL3 transcripts using RNA isolated from the hepatopancreas and performed using a total of 2 mg RNA isolated from muscle (lane 1 & 6),
muscle of the Pacific abalone Haliotis discus hannai. RNA was isolated mixtures containing various ratios ranging from 3:1 (lanes 2 & 7), 1:1
from the hepatopancreas (lanes 1–12) and muscle (lanes 13–16) using (lanes 3 & 8), to 1:3 (lanes 4 & 9) of muscle and hepatopancreatic RNA and
different isolation methods: I (lanes 1–3 & 13), II (lanes 4–6 & 14), and III RNA isolated from the hepatopancreas (lanes 5 & 10). RNA isolated from
(lanes 7–12, 15, & 16) as indicated. The same amounts (2 mg) of RNA were the hepatopancreas using methods III (lanes 2–5) and I (lanes 6–10) was
used for cDNA synthesis followed by PCR analysis. The sizes (kb) of the used. Polymerase chain reaction was carried out with each cDNA template
molecular weight markers (M) are indicated. under the same conditions.
RNA ISOLATION FROM ABALONE TISSUES 203

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

The development of molecular techniques that can be


applied to abalone is important for understanding and character-
izing its growth-related phenotypes (Chen et al. 2006, Lucas et al.

Figure 5. Levels of cDNA synthesized from reverse transcription as


determined by the fluorescence of Cy5-dCTP incorporated into cDNA Figure 6. Competitive PCR analysis using different ratios of cDNA
products. Competitive reverse transcription was performed with a total of templates prepared from RNA isolated from the hepatopancreas of the
2 mg of RNA isolated from muscle (columns 1 & 6) alone, mixtures Pacific abalone Haliotis discus hannai using methods I and III, re-
containing various ratios, i.e., 7.5:2.5 (columns 2 & 7), 5:5 (columns 3 & 8), spectively. Polymerase chain reaction was performed using 10 mL cDNA
and 2.5:7.5 (columns 4 & 9), of muscle and hepatopancreatic RNA, and mixtures containing 10:0 (lane 1 & 8), 9:1 (lane 2 & 9), 7:3 (lane 3 & 10),
RNA isolated from the hepatopancreas (columns 5 & 10). RNA isolated 5:5 (lane 4 & 11), 3:7 (lane 5 & 12), 1:9 (lane 6 & 13), and 0:10 (lane 7 &
from the hepatopancreas using methods III (A) and I (B) was used. Levels 14) ratio mixtures of cDNA prepared from hepatopancreatic RNA
of fluorescence incorporated into the cDNA were compared with that isolated using methods III and I (lanes 1–7). In lanes 8–14, PCR was
prepared from muscle RNA set as 100%. Values represent means % SE of performed using the same ratios of cDNA mixtures but containing the
triplicate experiments, and different superscripts indicate significant same amount of water instead of cDNA templates prepared from
differences (P < 0.05). hepatopancreas RNA using method I.
204 CHOI ET AL.

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.

LITERATURE CITED
Apt, K. E., S. K. Clendennen, D. A. Powers & A. R. Grossman. 1995. Kansal, R., K. Kuhar, I. Verma, R. N. Gupta, V. K. Gupta & K. R.
The gene family encoding the fucoxanthin chlorophyll proteins from Koundal. 2008. Improved and convenient method of RNA isolation
the brown alga Macrocystis pyrifera. Mol. Gen. Genet. 20:455–464. from polyphenols and polysaccharide rich plant tissues. Indian J.
Atmar, R. L., T. G. Metcalf, F. H. Neill & M. K. Estes. 1993. Detection Exp. Biol. 46:842–845.
of enteric viruses in oysters by using the polymerase chain reaction. Li, Z. & H. N. Trick. 2005. Rapid method for high-quality RNA
Appl. Environ. Microbiol. 59:631–635. isolation from seed endosperm containing high levels of starch.
Atmar, R. L., F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Biotechniques 38:872–876.
Woodley, T. G. Metcalf & M. K. Estes. 1995. Detection of Norwalk Li, Y., W. Wang, X. Du & Q. Yuan. 2010. An improved RNA isolation
virus and hepatitis A virus in shellfish tissues with the PCR. Appl. method for filamentous fungus Blakeslea trispora rich in poly-
Environ. Microbiol. 61:3014–3018. saccharides. Appl. Biochem. Biotechnol. 160:322–327.
Baranski, M., M. Rourke, S. Loughnan, B. Hayes, C. Austin & N. Liu, X., X. Liu & G. Zhang. 2007. Identification of quantitative trait loci
Robinson. 2008. Detection of QTL for growth rate in the blacklip for growth-related traits in the Pacific abalone Haliotis discus hannai
abalone (Haliotis rubra Leach) using selective DNA pooling. Anim. Ino. Aquacult. Res. 38:789–797.
Genet. 39:606–614. Lopez, L. M., P. Tyler & M. T. Viana. 1998. The effect of temperature
Barlow, J. J., A. P. Mathias, R. Williamson & D. B. Gammack. 1963. A and artificial diets on growth rates of juvenile Haliotis tuberculata
simple method for the quantitative isolation of undegraded high (Linnaeus, 1758). J. Shellfish Res. 17:657–662.
molecular weight ribonucleic acid. Biochem. Biophys. Res. Commun. Lucas, T., M. Macbeth, S. M. Degnan, W. Knibb & B. M. Degnan. 2006.
13:61–66. Heritability estimates for growth in the tropical abalone Haliotis asinina
Britz, J. P., T. Hecht, J. Knauer & M. G. Dixon. 1994. The development using microsatellites to assign parentage. Aquaculture 259:146–152.
of an artificial feed for abalone farming. S. Afr. J. Sci. 90:7–8. MacKay, R. M. & J. W. Gallant. 1991. Beta-tubulins are encoded by at
Brunet, M., J. Arnaud & J. Mazza. 1994. Gut structure and digestive least four genes in the brown alga Ectocarpus variabilis. Plant Mol.
cellular processes in marine Crustacea. Oceanogr. Mar. Biol. Annu. Biol. 17:487–492.
Rev. 32:335–367. Manickavelu, A., K. Kambara, K. Mishina & T. Koba. 2007. An
Bugos, R. C., V. L. Chiang, X. H. Zhang, E. R. Campbell, G. K. Podila efficient method for purifying high quality RNA from wheat pistils.
& W. H. Campbell. 1995. RNA isolation from plant tissues re- Colloids Surf. B Biointerfaces 54:254–258.
calcitrant to extraction in guanidine. Biotechniques 19:734–737. McGaw, I. J. & D. L. Curtis. 2013. A review of gastric processing in
Cathala, G., J. F. Savouret, B. Mendez, B. L. West, M. Karin, J. A. decapod crustaceans. J. Comp. Physiol. B 183:443–465.
Martial & J. D. Baxter. 1983. A method for isolation of intact, Nelson, M. M., D. L. Leighton, C. F. Phleger & P. D. Nichols. 2002.
translationally active ribonucleic acid. DNA 2:329–335. Comparison of growth and lipid composition in the green abalone,
Chen, H. L., H. S. Yang, R. Huang & H. J. Tsai. 2006. Transfer of Haliotis fulgens, provided specific macroalgal diets. Comp. Biochem.
a foreign gene to Japanese abalone (Haliotis diversicolor supertexta) Physiol. B 131:695–712.
by direct testis-injection. Aquaculture 253:249–258. Richards, G. P. 1999. Limitations of molecular biological techniques for
Choi, M. J., G. D. Kim, J. M. Kim & H. K. Lim. 2015. Differentially- assessing the virological safety of foods. J. Food Prot. 62:691–697.
expressed genes associated with faster growth of the Pacific abalone, Rossen, L., P. Nørskov, K. Holmstrøm & O. F. Rasmussen. 1992.
Haliotis discus hannai. Int. J. Mol. Sci. 16:27520–27534. Inhibition of PCR by components of food samples, microbial
Chomczynski, P. & N. Sacchi. 1987. Single-step method of RNA diagnostic assays and DNA-extraction solutions. Int. J. Food
isolation by acid guanidinium thiocyanate-phenol-chloroform ex- Microbiol. 17:37–45.
traction. Anal. Biochem. 162:156–159. Salzman, R. A., T. Fujita, K. Zhu-Salzman, P. M. Hasegawa & R. A.
Dellacorte, C. 1994. Isolation of nucleic acids from the sea anemone Bressan. 1999. An improved RNA isolation method for plant tissues
Condylactis gigantea (Cnidaria: Anthozoa). Tissue Cell 26:613–619. containing high levels of phenolic compounds or carbohydrates.
El-Maklizi, M. A., A. Ouf, A. Ferreira, S. Hedar & E. Cruz-Rivera. Plant Mol. Biol. Report. 17:11–17.
2014. A localized PCR inhibitor in a porcelain crab suggests Sambrook, J. & D. W. Russell. 2001. Molecular cloning-laboratory
a protective role. PeerJ 2:e689. manual, 3rd edition. Cold Spring Harbor, NY: Cold Spring Harbor
Floreto, E. A., S. Teshima & S. Koshio. 1996. The effects of seaweed Laboratory Press. pp. A8.14–A8.17.
diets on the lipid and fatty acids of the Japanese disc abalone To, K. Y. 2000. Identification of differential gene expression by high
Haliotis discus hannai. Fish. Sci. 62:582–588. throughput analysis. Comb. Chem. High Throughput Screen. 3:235–241.
Gao, J., J. Liu, B. Li & Z. Li. 2001. Isolation and purification of van der Merwe, M., P. Franchini & R. Roodt-Wilding. 2011. Differen-
functional total RNA from blue-grained wheat endosperm tissues tial growth-related gene expression in abalone. Mar. Biotechnol.
containing high levels of starches and flavonoids. Plant Mol. Biol. (NY) 13:1125–1139.
Report. 19:185a–185i. Vanessa, D. R. F., P. T. Angela, C. O. Mariana & C. Pio. 2008. RNA
Hahn, K. O. 1989. Nutrition and growth of abalone. In: Handbook of isolation method for polysaccharide rich algae: agar producing
culture of abalone and other marine gastropods. Boca Raton, FL: Gracilaria tenuistipitata (Rhodophyta). J. Appl. Phycol. 20:9–12.
CRC Press. pp. 135–180. White, E. J., M. Venter, N. F. Hiten & J. T. Burger. 2008. Modified
Hong, Y. K., S. D. Kim, M. Polne-Fuller & A. Gibor. 1995. DNA cetyltrimethylammonium bromide method improves robustness and
extraction conditions from Porphyra perforata using LiCl. J. Appl. versatility: the benchmark for plant RNA extraction. Biotechnol. J.
Phycol. 7:101–107. 3:1424–1428.

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