A method for RNA isolation from marine macro-algae

ANALYTICALBIOCHEMISTRY

174,650-657(1988)

A Method for RNA Isolation from Marine Macro-Algae XING Department

of Biological

Sciences,

University

Su AND

AHARON

ofCal$ornia

GIBOR

at Santa

Barbara,

Santa

Barbara,

California

93106

Received January 8, 1988 Sulfated, carboxylic polysaccharides and polyphenols found in marine macro-algae interfere with RNA isolation from these plants and inhibit RNA activities in vitro. Methods based on differential precipitation of RNA or carbohydrates in high salts were used to eliminate the acidic carbohydrates. To protect RNA from inactivation by oxidized polyphenols, strong reducing reagents were used to prevent polyphenol oxidation. RNA was successfully isolated from Macrocystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). mRNA isolated from the total RNA was shown to be translationally active. o 1988 Academic Press, Inc.

KEY WORDS: RNA isolation; algal RNA; marine pha; acidic carbohydrates; sulfated polysaccharides;

Marine algae are an important group of photosynthetic plants. Studies on the molecular biology of these organisms have been limited to unicellular algae (l-4), with little work done on macro-algae (5). One of the limiting factors is the difficulty of RNA isolation from marine macro-algae due to the presence of acidic polysaccharides and polyphenols. Acidic polysaccharides, i.e., sulfated polysaccharides (e.g., agar, carrageenan, and fucan) and carboxylic polysaccharides (e.g., alginic acid), are the major carbohydrates in red and brown algae (6,7). These polymers have densities and ion binding properties similar to those of RNA. They are more water soluble than the neutral polysaccharides of land plants (8) and their solutions are highly viscous. Polyphenols are another component of macro-algae. They bind to macromolecules by hydrogen bonding or by covalent bonding when quinones form upon oxidation (9,lO). Thus they are potentially strong inhibitors of RNA activities. The presence of these substances makes it difficult to isolate RNA directly from tissue lysates of marine macro-algae by standard CsCl density gradient centrifugation ( 11,12) or LiCl precipitation (13-16). 0003-2697188

$3.00

Copyri&t 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

algae; Macrocystis; Porphyra; Enteromorcarboxylic polysaccharides; polyphenols.

To isolate biologically active RNA from marine macro-algae, special techniques are needed to eliminate acidic polysaccharides and polyphenols. In the following, we describe a technique, which has been successfully used to isolate translationally active RNA from Macrocystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). MATERIALS

AND METHODS

Materials

All reagents used were analytical grade purchased from Sigma Co. unless otherwise specified. Labware and solutions were prepared as suggested ( 12). Fresh tissues of M. pyrifera (Phaeophyta), P. schizophylla (Rhodophyta), and E. intestinalis (Chlorophyra) were collected locally (Santa Barbara, CA). The tissues were blotted dry, cut into small pieces, and then powdered in liquid nitrogen with a mortar and pestle. The powders were stored frozen at -70°C. Total RNA Isolation Procedure

In the following procedure, the term chloroform refers to a mixture of chloroform and isoamyl alcohol at 49:l; phenol refers to a 650

RNA ISOLATION

FROM MARINE

7Krs Brown

stage

algae

Homogenate

Red

in

651

MACRO-ALGAE

or

green

A

algae

Tris-Borate

Ethanol+K+

\

~ge-,:::,l:n::y-Hc'

Chloroform

::::;

extraction

(A.3)

Clea>hokgenate LiC1+2-mercaptoethanol 11 Crude/RNA\pellet brown

algae RNA

Red J Tris-Borate

I"

\

RNA

in

Ethanol+K+

or

green

algae

Tris-HCl

Stage

B

(B.1)

K+ Phenol

extraction

(8.2) I

I Chloroform

extractlon

NaBH4

(B.3) LiCl

(B.4)

i/

RNA pellet I I RNA III T;is-HCl buffer I I Ca++-RNA preclpltatlon RNA

III

I

! EDTA

Stage (C.1)

buffer

CC.2)

Phenol

extraction

Chloroform

extraction

Ethanol Purified FIG.

C

(C.3)

RNA

1. Total cellular RNA isolation scheme.

mixture of phenol and chloroform at 1: 1. Solvent extractions were done by vigorous shaking of the aqueous solutions with indicated organic solvents for l-2 min, followed by centrifugation at about 20,OOOg for 5- 10 min. The complete procedure was divided into three stages which are outlined in Fig. 1 and detailed in the following.

sue) for 2 min in lysing buffer (2% SDS,’ 1% 2-mercaptoethanol, 50 mM EDTA, and 150 mM Tris-borate, pH 7.5) in a Bead-Beater (Biospec Products, USA). For red and green algae, Tris-borate was replaced by Tris-HCl. (iia) SDS-guanidinium precipitation for red and green algae). A l/10 bufer volume of 8 M guanidinium-HCI was added to the homogenate to precipitate SDS, proteins, and some large polysaccharides. The mixture was

Stage A (i) Tissue homogenization. Powdered tissues were homogenized (2-3 ml buffer/g tis-

I Abbreviation

used: SDS, sodium dodecyl sulfate.

652

SU AND

shaken vigorously for 1 min, followed by chloroform extraction. (iib) Ethanol-potassium (K’) precipitation lfor brown algae). The homogenate was mixed quickly with a buffer volume of 100% ethanol and then with 4 buffer volume of 5 M potassium acetate (K+). Mixing was continued for 1 min and followed by chloroform extraction. (iii) LiCl precipitation, The recovered aqueous phase was extracted once with phenol and once with chloroform. RNA in the aqueous solution was then precipitated by 3 M LiCl (i vol of 12 M LiCl). 2-Mercaptoethano1 was added to 1% (v/v) to prevent polypheno1 oxidation. The solution was kept at about - 10°C overnight. Stage B (i) Solubilizing RNA. RNA was collected by centrifugation at about 20,OOOgfor 90 min at 4°C. The pellets were dissolved in about l/ 50 of the original volume of 0.5X lysing buffer (see Stage A. I). For brown algae, Stage A.2b (ethanol-K+ precipitation) was repeated once; for other algae, K+ was added to 0.5 M, followed by chloroform extraction. {ii) Phenol and chloroform extraction. Phenol extraction was carried out for two or three times. The solution was then extracted with chloroform. Chloroform extraction was repeated once or twice if there was any interphase. (iii) NaBH, reduction for brown or polyphenol-containing algae). Solid NaBH4 was added, with gentle mixing, to the RNA solution to a final concentration of about 1%. Oxidized compounds (e.g., quinones) were reduced by NaBH4. Since a large number of bubbles could be generated, large containers were used ( 10 times the solution volume). (iv) LiCl precipitation. After the bubbles had dissipated, 2 vol of HZ0 and 1 vol of 12 M LiCl were added to the RNA solutions. 2Mercaptoethanol was added to about I % (v/ v) and the mixture was incubated at about - 10°C overnight.

GIBOR

Stage C (i) CaC12 precipitation. Precipitated RNA was collected by centrifugation (see Stage B. I). RNA pellets were dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at the same volume as in Stage B. 1. If the pellets were still colored, Stage B.3 was repeated. An equal volume of 1 M CaCIZ was then added to precipitate RNA, leaving neutral compounds (polyphenols) in solution. RNA precipitation formed on ice within 30 min. (ii) Phenol extraction. Ca’+-precipitated RNA was collected by centrifugation (20,OOOg for 15 min) and dissolved in a buffer containing 10 mM Tris-HCl and 20 mM EDTA. After the RNA pellets had dissolved completely, a vol of 5 M NaCl and an equal volume of phenol were added. Phenol extraction and chloroform extraction were repeated once or twice. (iii) Ethanol precipitation. Ethanol (2.5 vol of 100%) was added to tha aqueous solutions to precipitate RNA. RNA in ethanol was then stored at -20°C. mRNA Isolation and Its Translation in Vitro (i) Cellulose chromatography. The total cellular RNA pellet after ethanol precipitation was dissolved in TE buffer (less than 1 mg RNA/ml). NaCl concentration was adjusted to 0.5 M and SDS to 0.1%. The solution was passed three times through a cellulose column which had been sterilized with 0.2 M NaOH and equilibrated with column buffer (10 mM Tris-HCl, pH 7.5, 1 mrvr EDTA, 0.5 M NaCl, and 0.1% SDS). RNA was not retained by the column and remained in solution. The column was finally washed with 4 bed vol of column buffer. All the passed solutions containing RNA were combined. (ii) Oligo(dT)-cellulose chromatography. mRNA was isolated by loading the above RNA solution onto an oligo(dT)-cellulose column following standard procedures ( 17). The column was washed with column buffer and 0.1 M NaCl before mRNA was eluted with water. The eluted mRNA was brought

RNA ISOLATION

FROM MARINE

to 0.5 M NaCI, precipitated with 2.5 vol of ethanol, and stored at -20°C. (iii) Translational assay. mRNA was translated in vitro in a rabbit reticulocyte lysate system (Promega Biotec). mRNA (0.020.5 pg) in 2 ~1 H20, 1 ~1 [35S]methionine (1450 Ci/mmol, Amersham), and 7 ~1 lysate were combined and incubated at 30°C for 60 min. The efficiency of mRNA translation was determined by comparing the radioactivity incorporated into translated products of marine macro-algal mRNAs with those of equal amounts of Ch~amydomonas mRNA. RNA Quantitation

and Recovery Assay

RNA concentration was determined by uv absorption, assuming 1 unit of absorbance at 260 nm equals 50 pg of RNA/ml. mRNA concentration was estimated on an ethidium bromide plate by comparison with serially diluted RNA of known concentration ( 18). To estimate recovery of RNA, known amounts of RNA purified from Chlamydomonas were added to the macro-algal extracts (Stage A. 1). The amount of RNA recovered after the isolation procedure was determined for each sample. The recovery efficiency was calculated from data of three assays by the following equation: (X + A)E = R, where X is RNA in the extract (unknown); A is added RNA; E is efficiency of RNA recovery; and R is recovered RNA. Carbohydrate Estimation

and Polyphenol Content

Carbohydrate contents in RNA samples were determined by the phenol method ( 19). Ultraviolet absorbance (A) of a RNA sample (in distilled water) was measured from 220 to 300 nm to estimate uv absorptive contaminants. The ratio ofA23,-,/A260was used for estimating polyphenol contamination (see Results and Discussion for details). Agarose Gel Electrophoresis About 10 pg of total cellular RNA from each sample was separated in a 2% agarose

653

MACRO-ALGAE

gel (room temperature) at 100 V of constant voltage under nondenatured conditions. The gel contained ethidium bromide (1 pg/ml) and was prepared with TBE buffer (50 mM Tris-borate, pH 8.2, 1 mM EDTA). Polyacrylamide Gel Electrophoresis and Autoradiography Translation products were electrophoresed in a 9-12% gradient polyacrylamide gel (20) at 25 mA. The gel was treated by En3Hance (NEN Products), dried in a gel dryer (BioRad), and exposed to XAR-5 film (Kodak). RESULTS

AND

DISCUSSION

Eficiency of Carbohydrate

Removal

The principle used in this procedure to separate RNA from carbohydrates is that solubilities of RNA and acidic carbohydrates differ in salt solutions under selected conditions. Carbohydrates of M. pyrifera extracts were readily precipitated in the presence of 20% ethanol and 0.5 M K”. Under these conditions, RNA remained in solution. Either 20% ethanol or 0.5 M K+ alone could not precipitate the carbohydrates of the brown alga; instead, K+ caused gelation of the carbohydrates and made it more difficult to isolate RNA. The low concentration of ethanol appeared to inhibit gelation and to stimulate precipitation of the carbohydrates. The ethanol-K+ method, however, was not so effective on carbohydrates of red (P. schizophylla) and green (E. intestinalis) algae. For these algae, large polysaccharides could be only partially removed by precipitation with SDS and guanidinium-HCl. Several other steps were also important in the removal of carbohydrates. LiCl precipitated RNA and separated it from carbohydrates, DNA, and proteins. Some co-precipitated carbohydrates could be removed by phenol or chloroform extractions in the presence of high concentration of Kf or Naf ions. As shown in Table 1, the carbohydrates found in isolated RNA samples were at low levels. The degree of carbohydrate contami-

654

SU AND GIBOR TABLE 1

CHARAC~ERIZATIONOFALGAL RNA ISOLATEDFROM M.pyrifera M

C Carbohydrates (mg/mg RNA) PoMhenol&~/hd Recovery (%) Translation efficiency (%)

0.43 (k0.02)b ND 100

(M), P. schizophylla (P),AND E. intestinalis (E)"

0.17 0.38 67.5 56.5

(kO.02) (kO.02) (k4) (+19)

P 0.13 0.43 62.0 57.7

(kO.03) (kO.02) (k5) (k15)

E 0.37 (kO.02) 72.9 (+6) 67.4 (*IO)

Note. C, Chlamydomonas reinhardtii. -, Not detectable. ND, Not determined. ’ See text for details and explanation. ’ Determined from wheat germ RNA (Calbiochem).

nation was related to the carbohydrate composition of the plants. Contaminating carbohydrates were not detectable in RNA preparations from the green alga. Small amounts of carbohydrates were present in RNA preparations from the brown alga and the red alga. In these algae, the extracellular matrices consist mainly of acidic carbohydrates. Removal of Polyphenols Previous experiments showed that homogenates of brown alga were extremely sensitive to oxidizing reagents or oxygen. A brown color developed upon oxidation of the homogenate, known as the Browning Effect ( IO). It was also found that oxidized polyphenols (quinones) bound to RNA more readily than to carbohydrates. Two approaches were taken to minimize polyphenol contamination. A buffer containing a high concentration of borate was used in the early steps because borate forms H-bonded complexes with polyphenols (2 1,22). Reducing reagents were used to prevent oxidation of polyphenols or to reduce quinones. A high concentration of 2-mercaptoethanol was included in the buffers. It was important to add 2-mercaptoethanol to the LiCl solution when RNA was precipitated, otherwise polyphenol oxidation occurred during the long incubation time. NaBH, was a good reducing reagent of quinones, which was indicated by the decrease in brown color of RNA solutions after NaBH4 treatment. Once quinones were reduced back to polyphenols by NaBH,, they

could be separated from RNA by precipitating RNA with Li+ or Ca2+. To determine the degree of polyphenol contamination, the absorbances (A) of RNA samples were examined in the uv range. The ratio between readings at 230 and 260 nm (A230/A260) was 2 or more for polyphenols (quinones) (23) and about 0.43 for standard RNA (wheat germ RNA from Calbiochem). The A,,,/A,,, ratio shifted when polyphenols were present in RNA samples. For example, A230/A260 was 0.65 when equal amounts of polyphenols were added to the standard RNA sample. TheA230/A260 ratios ofthe purified algal RNA samples were 0.38 (M. pyrifera), 0.43 (P. schizophylla), and 0.37 (E. intestinalis), which were equal to or less than that of the standard RNA. The data suggest that very little polyphenol was present in the isolated algal RNA preparations. Integrity of Isolated RNA Algal RNAs were analyzed by agarose gel electrophoresis. As shown in Fig. 2, the size distributions ofthe RNAs were wide. Because about 99% of total RNA was rRNA, the integrity of a RNA sample was measured by the integrity of rRNA. Intact Chiamydomonas rRNAs gave bands corresponding to 25S, 23S, ISS, 16S, and some species of small RNA (lane C of Fig. 2). On the other hand, degraded RNA produced smeared patterns (data not shown). Chlarnydomonas RNA recovered from algal extracts showed basically the same band pattern as the control RNA

RNA ISOLATION

FROM MARINE

PMEC

-25s -23s -18s -16s

-tRNA

FIG. 2. RNA agarose gel electrophoresis. Total RNA (10 pg) from each alga was fractionated by electrophoresis in a 2% agarose gel prepared with TBE buffer (see Materials and Methods). RNA was visualized by staining with ethidium bromide. P, M, E, and C represent RNAs from P. schizophylla, M. pyrifera, E. intestinalis, and Chlamydomonas reinhardtii, respectively. The sizes of C. reinhardtii rRNAs are marked.

(lane C of Fig. 2). If large amounts of RNA were degraded, smeared patterns should be observed. Distinct bands were present in all of the algal RNA samples, suggesting that RNA degradation was not significant during RNA isolation. It appeared that some degree of specific degradation did occur. Theoretically, the mass ratio of 25s RNA to 18s RNA should be about 2:l. As seen in Fig. 2, this was not true in the algal RNA samples. The 18s RNA band appeared more intense than the 25s band. There were also two or three major bands of unknown origin below the 18s RNA. These lower bands may represent cleavage products of 25s RNA since 28s (25s) rRNA of some other organisms is known to contain some sensitive cleavage sites. Once broken, the 28s (25s) rRNA produced several unique sizes of RNA below 20s (24-28). Eficiency

of RNA Recovery

RNA recovery assays were done with the three algae and the RNA isolation procedure

MACRO-ALGAE

655

was designed to maximize the recovery efficiencies. As shown in Table 1, about twothirds of the Chlamydomonas RNA added to the algal extracts could be recovered by this procedure. It was found that compounds which readily formed H-bonds with RNA were potential inhibitors of RNA precipitation. If the Tris-borate concentration in buffers was higher than 0.2 M, it reduced RNA recovery. It was not the case for TrisHCl buffer. Guanidinium also inhibited RNA precipitation at concentrations higher than 0.5 M. However, high concentrations of Kt and Na+ facilitated RNA precipitation. Thus, to prevent RNA precipitation during phenol or chloroform extraction, Na+ and K+ salts at concentrations higher than those indicated should be avoided. The highest yields obtained from P. schizophylla and M. pyrifera were 1.6 and 0.9 mg/ 10 g tissue, respectively. The yields depended on plants used, physiological conditions of the tissue when collected, and percentage of cells which had been opened during homogenization. In some red algal tissues, less than half of the cells could be broken, thus greatly reducing the yield. Eficiency of mRNA Translation in Vitro

mRNA isolated from the three algae were all translatable in a rabbit reticulocyte lysate system (Fig. 3). The sizes of translated proteins were up to or more than 100 kDa. This implied that unusual codons were not used in the species tested. The translation efficiencies of the three mRNA samples were 67.4 (E. intestinalis), 56.5 (M. pyrifera), and 57.7% (P. compared to the same schizophylla), amounts of mRNA isolated from Chlamydomonas (Table 1). mRNA from the brown and the red algae seemed to be more difficult to translate. This might be due to the presence of inhibitors. Cellulose chromatography was important for isolating active mRNA from brown and red algae. Cellulose chromatography was used before oligo(dT)-cellulose chromatography to remove contaminants which absorbed

SU AND GIBOR OME

P

kDa

the band patterns of translated proteins (data not shown), the inhibition by contaminants was probably due to partial inactivation of the translation system.

l16.3 925

66.2

31.0

Other Features of the Procedure

SDS was substituted for guanidinium isothiocyanate in lysing buffer to avoid the need for excessive dilution before RNA precipitation (29-32). Thus the technique has a small buffer-volume/tissue-weight ratio as compared to other commonly used methods (16). The procedure does not require ultracentrifugation. These features add to its convenience.

21.5

ACKNOWLEDGMENTS FIG. 3. Autoradiography of in vitro-translated algal proteins. Algal mRNAs were translated in a reticulocyte lysate system. The protein products were electrophoresed in a 9- 12% polyacrylamide gel. The gel was dried and autoradiographed with XAR-5 film. M, E, and Prepresent translated products from mRNAs of corresponding algae (see legend of Fig. I). 0, Translation background (no mRNA added)

to the cellulose matrix at high salt concentrations. These contaminants, which could be eluted from the cellulose column by low salt buffer, were found to inhibit translation when they were added to Chlamydomonas mRNA (data not shown). The isolated mRNA appeared to still contain substances which inhibited translation in their oxidized state. Although polyphenols were hardly detectable in these RNA samples, traces of them could have a strong inhibiting effect on translation. When mRNA isolated from P. schizophylla was treated by 0.5% (w/v) NaI04 for 15 min, translation activity of the mRNA was inhibited but Chlatreated by the same mydomonas mRNA method was still fully active (data not shown). Since contaminating carbohydrates were still present in brown and red algal RNA, they might also inhibit translation. Because translation efficiency was dependent on the dose of mRNA used and inhibition did not change

The authors thank Dr. M. Polne-Fuller for providing the P. schizoph@ tissues and Dr. D. Kaska for helpful suggestions on the manuscript. This work was supported by a grant from the NSF (Grant No. DCB 95-10326) and a grant from the Biotechnology Research and Education Program of the University of California. REFERENCES 1. Li-Weber, M.. and Schweiger, H. G. (1985) Eur. J. Cell Biol. 41(2), 419-428. 2. Hinnebusch. A. G., Klotz, L. C., Immergut, E., and Loeblich, A. R. III. (1980) Biochemistry 19, 1744-1755. 3.

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RNA ISOLATION

FROM MARINE

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