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Research Article
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road,
Honolulu, HI 96822, United States.
It is difficult to extract high-quality RNA of sufficient quantity from the stem tissues of tree
legumes, such as Acacia koa and Leucaena leucocephala, because they contain high amounts
of phenolic compounds and polysaccharides. The objective of this study was to develop an
improved protocol that produces high-quality RNA from the stem tissues of these tree legumes.
We developed a modified method that utilizes a lysis buffer containing a mixture of two
commercially available reagents, RLT buffer from Qiagen RNeasy Kit and Fruit-mate from
Takara. Comparison of the modified method with four other RNA extraction methods (Qiagen
RNeasy, Fruit-mate, TRIzol, and cetyltrimethylammonium bromide (CTAB)) showed that the
modified method was far superior to the other methods. The extracted RNA produced
reproducible DNA products in reverse transcription-polymerase chain reaction (RT-PCR). This
improved protocol is rapid and easy, and it will facilitate genetic studies of A. koa, L.
leucocephala, and other woody plants.
Keywords: RNA extraction, Acacia koa, Leucaena leucocephala, tropical tree legumes, Hawaii
INTRODUCTION
Acacia koa is a leguminous timber wood tree
endemic to the Hawaiian Islands. With its beautiful
texture, hardiness, and carving quality of the wood, it is
the most economically important tree in Hawaii (Baker et
al., 2009; Yanagida et al., 2004). Similarly, Leucaena
leucocephala is a tree legume, which is widely used for
agroforestry (Brewbaker et al., 1990). For genetic studies
of A. koa and L. leucocephala using molecular biology
techniques such as complementary DNA (cDNA) library
construction, reverse transcription-polymerase chain
reactions (RT-PCR), and microarray analysis, it is a
critical step to extract high-quality RNA in sufficient
quantity. The isolation of RNA from these woody species
poses a particular challenge in that they often contain
high levels of polysaccharides and phenolic compounds,
which can co-purify with RNA and inhibit enzymatic
manipulations (Loomis, 1974; Nassuth et al., 2000;
Wilkins et al., 1996). Many existing protocols for plant
Ishihara et al.
031
032
Table 1. Quantity and quality of total RNA extracted by five methods from woody stems of A. koa and L.
leucocephala (mean SE) (n=3)
Extraction
Method
A. koa
Modified
RNeasy
Fruit-mate
TRIzol
CTAB
L. leucocephala
Modified
RNeasy
Fruit-mate
TRIzol
CTAB
Quantity
(g/g plant)
A260/A280 Ratio
A260/A230 Ratio
RIN
213.2 33.9
102.1 5.0
40.4 3.5
81.8 14.8
16.6 3.8
2.14 0.01
1.98 0.01
1.79 0.01
1.58 0.07
1.83 0.03
2.09 0.15
0.94 0.02
0.28 0.03
0.32 0.02
1.85 0.20
8.0 0.0
6.8 0.1
3.0 0.6
2.6 0.0
ND*
206.7 24.5
19.4 6.04
275.3 16.7
129.1 18.6
831.2 47.2
2.14 0.00
2.09 0.05
0.28 0.01
1.58 0.03
1.85 0.01
2.11 0.02
0.70 0.06
0.97 0.06
0.32 0.06
2.29 0.02
6.8 0.1
ND*
5.2 0.1
ND*
4.5 0.9
* indicates that RIN value was not detectable due to the RNA concentrations being too low. indicates significant
difference (p <0.05) with the modified protocol. indicates significant difference (p <0.01) with the modified protocol.
method
Ishihara et al.
033
Figure 1. Gel-like image of total RNA obtained from Agilent Bioanalyzer. Total RNA of A. koa, extracted by (lane 1) the
modified method, (lane 2) the Qiagen RNeasy method, (lane 3) the Fruit-mate method, (lane 4) the TRIzol method, and
(lane 5) the CTAB method. Total RNA of L. leucocephala, extracted by (lane 6) the modified method, (lane 7) the Qiagen
RNeasy method, (lane 8) the Fruit-mate method, (lane 9) the TRIzol method, (lane 10) the CTAB method. M - a marker.
034
Figure 2. RT-PCR analysis of actin gene fragment. No-RT-PCR controls with RNA from (A) A. koa and (B) L. leucocephala
and RT-PCR of RNA from (C) A. koa and (D) L. leucocephala. RNA used was extracted by (lane 1) the modified method, (lane 2)
the Qiagen RNeasy method, (lane 3) the Fruit-mate method, (lane 4) the TRIzol method, and (lane 5) the CTAB method. M - a
1 kb DNA marker.
ACKNOWLEDGMENTS
We would like to acknowledge Tyler Jones and
Nick Dudley from HARC and Michael Honda for providing
A. koa and L. leucocephala seeds. We would also like to
acknowledge Bradley Porter for his help. The research on
A. koa was supported by the McIntire-Stennis Grant
HAW00597-M. The research on L. leucocephala was
funded by the HATCH Project HAW05029-H. KI is
supported by a Monsanto Graduate Fellowship.
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