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Journal of Experimental Botany, Vol. 52, No. 360, pp. 1575-1579, July 1, 2001
© 2001 Oxford University Press

An efficient method for extraction of RNA from rice leaves at different ages using benzyl chloride

Yuji Suzuki, Amane Makino and Tadahiko Mae1

Department of Applied Plant Science, Graduate School of Agricultural Sciences, Tohoku University, Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

Received 18 December 2000; Accepted 19 March 2001


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
With a conventional method of RNA extraction using an acid guanidinium thiocyanate–water-saturated phenol–chloroform mixture, extraction efficiency of extractable RNA to total RNA (extractable RNA+ residual RNA) in rice leaves at various ages was 54–69%. With a new method, an improvement of the above, using benzyl chloride instead of water-saturated phenol together with further maceration with a small amount of quartz sand, the efficiency was increased to 81–95%. When RNA fractions obtained with the improved method were subjected to agarose gel electrophoresis, intact bands of 25 S and 17 S rRNAs were detected. With a DNA probe for rice rbcS, only a single band was observed on the blotted membrane. These results indicate that the improved extraction method of RNA with benzyl chloride is useful for quantitative and qualitative analysis of RNA in plant tissues such as stiff leaves of rice.

Key words: Oryza sativa L., RNA extraction, senescence.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
In physiological and biochemical studies in plants, it often becomes important to examine changes in levels of total RNA or mRNA of a particular gene per unit fresh weight or unit leaf area. In most previous studies, however, extraction efficiency of RNA from plant tissues and/or degradation and loss of RNA during preparation have not been examined. Therefore, it is unclear whether changes in the levels of RNA or mRNA were due to changes in their absolute amounts or to their extraction efficiency and/or degradation and loss during preparation, especially when the levels were compared among different tissues or the same tissues at different ages.

In this paper special attention was paid to extraction efficiency and recovery of RNA from rice leaves at different leaf ages. Hardening of leaf tissues proceeds with leaf development. Some nucleases are induced during leaf senescence (for a review see Dangl et al., 2000Go). Secondary metabolites such as polyphenols accumulate during senescence. These factors might affect the extraction efficiency and/or recovery of RNA from leaf tissues. Two methods of RNA extraction were compared. One is a conventional method using an acid guanidinium thiocyanate–water-saturated phenol–chloroform mixture (AGPC method) (Chomczynski and Sacchi, 1987Go). The other is a new method (BC method), an improvement of the above, which employs benzyl chloride instead of water-saturated phenol, together with further maceration with a small amount of quartz sand. Benzyl chloride is now commonly used for the extraction of genomic DNAs because it can destroy cell walls of plants, fungi and bacteria through its reaction with –OH residues in polysaccharides, including cellulose, hemicellulose, etc. Furthermore, similar to phenol, benzyl chloride can also extract proteins and other cell debris from the aqueous phase (Zhu et al., 1993Go).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Plant materials
Rice (Oryza sativa L. cv. Notohikari) was grown hydroponically in a greenhouse. The basal hydroponic solution used was the same as that described previously (Mae and Ohira, 1981Go). The solution was renewed once a week and its pH was adjusted to 5.5 with 1 N HCl. Whole leaf blades of expanding, mature and senescing leaves at the early stage and at the advanced stage were collected as samples. These leaves were collected between 11.00 h and 12.00 h. Leaves were immediately weighed and frozen in liquid nitrogen after cutting, and stored at -80 °C until analyses.

RNA extraction
The conventional method (AGPC method):
As a conventional method, the extraction method using an acid guanidinium thiocyanate–water-saturated phenol–chloroform mixture (Chomczynski and Sacchi, 1987Go) was employed with a slight modification. Frozen leaves were ground to a fine powder (for about 10 min) in a mortar with a pestle in the presence of liquid nitrogen in a cold room. Leaf powder was weighed and placed in denaturing solution (4.2 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% (w/v) sodium N-lauroyl sarcosine, and 5% (v/v) 2-mercaptoethanol) at a ratio of denaturing solution to leaves of 5–15 ml g-1 FW (15 ml g-1 FW for expanding leaves and 5 ml g-1 FW for senescing leaves). After transfer of 0.5 ml of the homogenate to a 2.0 ml disposable polypropylene tube, followed by the addition of 0.05 ml of 2 M sodium acetate, pH 4.0, and 0.5 ml of water-saturated phenol, the contents were mixed well. Then, 0.25 ml of a chloroform–isoamylalcohol mixture (24 : 1, v : v) was added and the tube was shaken for 15 s by hand. The tube was then kept on ice for 15 min, followed by centrifugation at 12 000 g at 4 °C for 15 min. After 0.4 ml of the aqueous phase was transferred to a 1.5 ml disposable polypropylene tube, it was mixed with 0.2 ml of 1.2 M sodium chloride and 0.8 M sodium citrate, and 0.2 ml of isopropanol to avoid the contamination of polysaccharides (Chomczynski and Mackey, 1995Go). The mixture was incubated at room temperature for 10 min, followed by centrifugation at 12 000 g at 4 °C for 15 min. The resulting RNA pellets were washed with 75% (v/v) ethanol, air-dried, and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

The improved method (BC method):
Frozen leaves were ground to a powder (for about 1–2 min) in a mortar with a pestle in the presence of liquid nitrogen in a cold room. Leaf powder was weighed and placed in denaturing solution as in the above AGPC method. The homogenate was further macerated with quartz sand (at a ratio of quartz sand to denaturing solution of 16.7–33.3 mg ml-1) in a mortar with a pestle in a draft chamber. Part (0.5 ml) of the homogenate was transferred to a 2.0 ml disposable polypropylene tube, followed by the addition of 0.25 ml of 3 M sodium acetate, pH 5.2, and 0.5 ml of benzyl chloride. After vigorous shaking at 60 °C for 30 min, 0.5 ml of a chloroform–isoamylalcohol mixture (24 : 1, v : v) was added to the tube, followed by shaking for 15 s by hand. Then the tube was kept on ice for 15 min, followed by centrifugation at 12 000 g at 4 °C for 15 min. After transfer of 0.5 ml of the aqueous phase to a 1.5 ml disposable polypropylene tube, it was mixed with 0.25 ml of 1.2 M sodium chloride and 0.8 M sodium citrate, and 0.25 ml of isopropanol. Treatment was then identical to the AGPC protocol from the step following addition of 0.2 ml of 1.2 M sodium chloride and 0.8 M sodium citrate, and 0.2 ml of isopropanol. Contamination of DNA was checked with Burton's method (Burton, 1956Go).

It is suspected that benzyl chloride has toxicological property as a carcinogen. The mixture including benzyl chloride should be carefully handled.

Quantification of extractable RNA in the aqueous phase
The amount of RNA in an aqueous solution was determined by absorbance at 260 nm (A260) with a spectrophotometer (UV-160A, Shimadzu, Kyoto, Japan). The amount of extractable RNA was calculated using the following equation:

where C is the amount of collected RNA, Vt is the total volume of the aqueous phase, and Vr is the volume of the recovered aqueous phase. For each sample, 5 µg of RNA was precipitated with ethanol and stored at –30 °C until required.

Extraction efficiency of RNA
Extraction efficiency was defined as the proportion of the amount of extractable RNA to the amount of total RNA (extractable RNA+residual RNA). To determine the amount of RNA remaining in the residual fraction (residual RNA), the residue after extraction of the extractable RNA was alkaline-digested according to Smillie and Krotkov with some modifications (Smillie and Krotkov, 1960Go). The residue was washed twice with 1.0 ml of 1-propanol, twice with 1.0 ml of methanol, three times with 0.5 ml of wash buffer (a half concentration of denaturing solution without 2-mercaptoethanol), twice with 1.0 ml of 5% (w/v) trichloroacetic acid, and twice with 1.0 ml of ethanol. The precipitates thus obtained were vacuum dried, suspended in 1.2 ml of 0.3 N KOH, and incubated at 37 °C for 16 h. After incubation, the solution was kept on ice for 15 min, followed by the addition of 0.06 ml of 6 N HCl and 0.12 ml of 60% (v/v) perchloric acid. The sample tube was placed on ice for a further 15 min and then subjected to centrifugation at 12 000 g at 4 °C for 15 min. The supernatant was transferred to a new 2.0 ml tube and frozen at -80 °C overnight. The content was then melted in the tube and centrifuged at 12 000 g at 4 °C for 15 min. A portion (0.2 ml) of the supernatant was transferred to a new 1.5 ml tube, and its pH was adjusted to 5.0 with 1 N KOH, followed by centrifugation at 12 000 g at 4 °C for 5 min.

A part (0.3 ml) of the above supernatant was mixed with 0.3 ml of 2xHPLC buffer (1xHPLC buffer=0.05 mM potassium phosphate, 8% (v/v) methanol, pH 4.4). The mixture was filtered through a 0.45 µm pore membrane of cellulose acetate (DISMIC-3CP, ADVANTEC, Tokyo, Japan). The filtrate was applied to HPLC (LC-10A, Shimadzu, Kyoto, Japan) attached to a silica gel-based reverse phase chromatography column (Mightysil RP-18 150–4.6 (5 µm), Kanto Chemical, Tokyo, Japan) and a guard column (Mightysil RP-18 5-4.6 (5 µm), Kanto Chemical, Tokyo, Japan) using 1xHPLC buffer as a solvent. Extractable RNA fractions obtained by the BC method or the AGPC method were alkaline-digested for HPLC analysis. The residual RNA contents were determined from the relative peak intensity of adenosine-2'(3')-monophosphate to that of extractable RNA.

Extraction efficiency was calculated with the following equation:

where E is extractable RNA and R is the residual RNA as described above.

Recovery of RNA
Recovery of RNA was determined from the loss of exogenously added RNA during preparation, caused by such factors as degradation of RNA and distribution of RNA other than that in the aqueous phase. A known amount of RNA (about 15–20 µg RNA) dissolved in 100 µl of TE buffer was added to a 2.0 ml tube containing 0.5 ml of the leaf homogenate after denaturation. RNA was then extracted according to the AGPC method or the BC method. Recovery was calculated with the following equation:

where E' is the amount of extractable RNA with addition of RNA, E is the amount of extractable RNA without addition of RNA, and A is the amount of RNA added.

Northern blot analysis
Five micrograms of the total RNA fraction was electrophoresed and blotted onto membrane according to the methods of Yamaya et al. (Yamaya et al., 1995Go), except for the use of positively charged nylon membrane (Hybond-N+, Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England) in this experiment.

The membrane was then prehybridized in 15 ml of high SDS hybridization buffer described in the DIG System User's Guide (Roche Diagnostics, IN, USA) at 53 °C for 2 h. The membrane was then hybridized with 15 ml of DIG-labelled DNA probe for rbcS at a concentration of 15 ng ml-1in the same hybridization buffer at 53 °C for 16 h. The probe was prepared from a fragment of rice rbcS gene (Matsuoka et al., 1988Go) with PCR DIG Probe Synthesis Kit (Roche Diagnostics, IN, USA) according to the manufacturer's instruction manual. The membrane was washed sequentially with 2xSSC that contained 0.1% (w/v) SDS at room temperature, twice for 10 min each, and then twice with 0.2xSSC that contained 0.1% (w/v) SDS at 68 °C for 20 min each. Chemiluminescence was detected with a DIG Luminescent Detection Kit (Roche Diagnostics, IN, USA) according to the DIG System User's Guide.

Determination of chlorophyll, nitrogen, and ribulose-1,5-bisphosphate carboxylase/oxygenase
Contents of chlorophyll, nitrogen, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were determined as indexes of leaf age, according to the methods of Makino and Osmond (Makino and Osmond, 1991Go).


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Leaf blades at different ages of rice plants were used throughout the experiments. Indexes of leaf age are shown in Table 1Go. The amounts of chlorophyll, Rubisco and total nitrogen per unit fresh weight were the largest in the mature leaves. All amounts decreased with the progress of leaf senescence. In advanced senescing leaves, the amounts of chlorophyll and Rubisco decreased to about one-fifth those in the mature leaves and the leaf tips started to dry up.


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  		Table 1. Chlorophyll, Rubisco, and nitrogen contents in rice leaves at different ages

Data are means±SE (n=3).

 
RNA extraction with conventional and improved methods
RNA was extracted from rice leaves at four different ages using two methods. One is a conventional method using water-saturated phenol (designated as the AGPC method) as an organic phase for separation of RNA from other components such as proteins. The other is a new method, an improvement of the above, using benzyl chloride instead of water-saturated phenol (designated as the BC method), together with further maceration with a small amount of quartz sand. The amount of extractable RNA per unit leaf fresh weight was considerably higher with the BC method than the AGPC method. By contrast, the amount of residual RNA was 2–5 times larger with the AGPC method than with the BC method, irrespective of leaf age (Table 2Go ). The amount of residual RNA with the AGPC method accounted for 31–46% of total RNA in the tissues. No difference was found for the total amount of RNA (extractable RNA+residual RNA) between the two methods in all leaves. The extraction efficiency of extractable RNA was calculated as 54–69% for the AGPC method and 81–95% for the BC method, respectively. The relative levels of RNA extracted from different tissues by each method were comparable. The BC method would be of most value when information about the absolute RNA level is required. Re-extraction of the interphase and lower phase from the chloroform/ phenol step with additional extraction buffer in the AGPC method affected the extraction efficiency in a minor way (data not shown). In advanced senescing leaves, the extraction efficiency was somewhat lower than that in other leaves.


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  		Table 2. Comparison of the amounts of extractable RNA and residual RNA and extraction efficiency between the BC method and the AGPC method in rice leaves at different ages

Data are means±SE (n=3).

 
The recovery of exogenously added RNA to samples was then examined (Table 3Go). The recovery was 91–105% with the BC method and about 90% with the AGPC method, indicating that degradation of RNA during isolation is not a serious factor affecting the yield of extractable RNA in the BC method.


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  		Table 3. Recovery of exogenously added RNA to leaf homogenates, followed by extraction with the AGPC method or the BC method from rice leaves at different ages

Data are means±SE (n=3).

 
The increase in the extraction efficiency by the BC method was due to the synergic effect of benzyl chloride treatment and further maceration with quartz sand. The yield of extractable RNA was not significantly increased with the BC method without maceration (data not shown). It has been reported that benzyl chloride destroys plant cell walls and improves the extractability of DNA from plant tissues (Zhu et al., 1993Go). However, use of benzyl chloride has not been reported. The results of this study clearly indicate that the use of hot benzyl chloride with further maceration of the leaf homogenate with quartz sand improves the yield of extractable RNA from leaf tissues as found for DNA. The AGPC method with maceration of the leaf homogenate with quartz sand, as in the BC method, did not improve the yield of extractable RNA (data not shown).

DNA contamination of the RNA fractions prepared by the BC method was not detected by Burton's method (Burton, 1956Go). DNA is thought to be distributed into the organic phase due to its hydrophobicity in acidic solution, as in the AGPC method. The A260/A280 and A260/A230 of the final RNA fractions were close to 2.0 and 2.0–2.4, respectively, in all samples, indicating little contamination of the RNA fraction by protein and polysaccharides.

Quality of extracted RNA
The RNA extracted with the BC method was electrophoresed on formaldehyde agarose gel and stained with ethidium bromide to check the integrity of the RNA (Fig. 1AGo). Intact bands of 25 S and 17 S rRNAs were detected on the gel in all RNA fractions from the leaves at different ages. There were also bands of low-molecular-weight RNAs (4–5 S) corresponding to tRNAs and rRNAs. The RNAs on the gel were subjected to Northern blot analysis (Fig. 1BGo). A single signal was detected by a DNA probe for rice rbcS. The signal intensity per loaded RNA was high in expanding and mature leaves, and extremely low in senescing leaves as reported previously (Jiang et al., 1993Go). This result indicates that RNA prepared by the BC method can be used for Northern blot analysis, too.



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  		Fig. 1. Gel electrophoresis and Northern blot analysis of RNA extracted with the BC method from rice leaves at different ages. (A) A portion (5 µg) of extracted RNA was loaded on 1% formaldehyde agarose gel and stained with ethidium bromide. RNA was extracted from young leaves (lanes 1), mature leaves (2), senescing leaves (3), and advanced senescing leaves (4). (B) The RNA was transferred to a positively charged nylon membrane and hybridized with DIG labelled DNA probe for rice rbcS. The lanes were the same as in (A).

 
Thus, the improved method for extraction of RNA using benzyl chloride achieves high extraction efficiency and recovery of RNA. This method would enable physiological studies to elucidate such things as the relationship between levels of a gene transcript and the levels of its encoding protein, especially during leaf development in which total RNA content dramatically changes.


   Acknowledgments
 
We thank Drs Ko Iba and Kensuke Kusumi (Kyushu University) for a kind gift of cDNA clones of rice and their advice on the experiment. We also thank Drs Tomoyuki Yamaya and Toshihiko Hayakawa and members of their laboratories for their instruction and fruitful discussion and for use of their equipment. This work was supported by Grants-in-Aid for Science Research 12460028 and for Science Research in Priority Areas 12025202 from the Ministry of Education, Science and Culture of Japan, by Grant-JSPS-RFTF 96L00604 for Research for the Future from the Japan Society for the Promotion of Science, and by the Bio Design Program (BDP-01-I-1-1) from the Ministry of Agriculture, Forestry and Fisheries, Japan.


   Notes
 
To whom correspondence should be addressed. Fax: +81 22 717 8765. E-mail: hikomae{at}biochem.tohoku.ac.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Burton K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical Journal 62, 315–322.[ISI][Medline]

Chomczynski P, Mackey K. 1995. Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 19, 942–945.[ISI][Medline]

Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162, 156–159.[ISI][Medline]

Dangl JL, Dietrich RA, Thomas H. 2000. Senescence and programmed cell death. In: Buchanan BB, Gruissem W, Jones RL, eds. Biochemistry and molecular biology of plants. Rockville: American Society of Plant Physiologists, 1044–1100.

Jiang CZ, Rodermel SR, Shibles RM. 1993. Photosynthesis, Rubisco activity and amount, and their regulation by transcription in senescing soybean leaves. Plant Physiology 101, 105–112.[Abstract]

Mae T, Ohira K. 1981. The remobilization of nitrogen related to leaf growth and senescence in rice plants (Oryza sativa L.). Plant and Cell Physiology 22, 1067–1074.[Abstract/Free Full Text]

Makino A, Osmond B. 1991. Effects of nitrogen nutrition on nitrogen partitioning between chloroplasts and mitochondria. Plant Physiology 96, 355–362.[Abstract/Free Full Text]

Matsuoka M, Kano-Murakami Y, Tanaka Y, Ozeki Y, Yamamoto N. 1988. Classification and nucleotide sequence of cDNA encoding the small subunit of ribulose-1, 5-bisphosphate carboxylase from rice. Plant and Cell Physiology 29, 1015–1022.[Abstract/Free Full Text]

Smillie RM, Krotkov G. 1960. The estimation of nucleic acids in some algae and higher plants. Canadian Journal of Botany 38, 31–49.

Yamaya T, Tanno H, Hirose N, Watanabe S, Hayakawa T. 1995. A supply on nitrogen causes increase in the level of NADH-dependent glutamate synthase protein and in the activity of the enzyme in roots of rice seedlings. Plant and Cell Physiology 36, 1197–1204.[Abstract/Free Full Text]

Zhu H, Qu F, Zhu LH. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Research 21, 5279–5280.[Free Full Text]


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