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Extraction and purification of total RNA from Sreptococcus mutans biofilms
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 365 (2007) 208–214 www.elsevier.com/locate/yabio
Extraction and purification of total RNA from Sreptococcus mutans biofilms Jaime A. Cury a, Hyun Koo b
b,*
a Faculty of Dentistry of Piracicaba, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil Eastman Department of Dentistry and Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14620, USA
Received 29 January 2007 Available online 24 March 2007
Abstract RNA isolation from Streptococcus mutans within biofilms is challenging because of the presence of extracellular polysaccharide matrix that interferes with RNA extraction procedures. In an effort to solve this difficult problem, we examined several protocols to extract and purify RNA from S. mutans biofilms. A combination of sonication (three times using a 30-s pulse at 7 W) with washing in phosphate-buffered saline removed most of the extracellular polysaccharides from the biofilms and provided the highest RNA yield. Further homogenization–mechanical cells disruption in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and acid phenol/chloroform yielded 547.2 ± 23.4 lg RNA/100 mg of biofilm dry weight. An additional acid phenol/chloroform extraction further improved the purification of RNA without significantly affecting the RNA yield. The combination of DNase I in silica gel-based column and recombinant DNase I in solution effectively removed the genomic DNA as determined by real-time quantitative reverse transcriptase PCR (RT–PCR), resulting in 92.0 ± 0.6 lg of purified RNA per 100 mg of biofilm dry weight. The complementary DNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results demonstrated a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for real-time RT–PCR analyses, and our data have relevance for isolation of RNA from other biofilm-forming microorganisms. Ó 2007 Elsevier Inc. All rights reserved. Keywords: RNA extraction; Real-time PCR; DNase I; Streptococcus mutans; Biofilms
Isolation of high-quality intact RNA with minimum levels of contaminants and in sufficient quantity is critical for downstream applications using functional genomics approaches, especially real-time quantitative reverse transcriptase PCR (qRT PCR)1 [1]. Real-time RT–PCR is a sensitive and reliable technique to measure gene expression but requires a reproducible and well-defined methodology for RNA extraction and purification for accurate determination of messenger RNA (mRNA) levels.
*
Corresponding author. Fax: +1 585 276 0190. E-mail address: [email protected] (H. Koo). 1 Abbreviations used: qRT–PCR, quantitative reverse transcriptase PCR; mRNA, messenger RNA; cDNA, complementary DNA; PBS, phosphatebuffered saline; ANOVA, analysis of variance; RIN, RNA integrity number. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.03.021
Several methods of RNA extraction and purification have been reported for bacteria grown in a planktonic state, including oral streptococci [2–7]. In contrast to planktonic cells, RNA isolation from microorganisms within biofilms is challenging because of the presence of an extracellular polysaccharide matrix that may interfere with the extraction and purification of the nucleic acids. RNA isolation and purification from polysaccharide-rich tissues, such as in plants and fruits, are difficult because these polymeric substances bind to nucleic acids, interfere with DNase activity, inhibit complementary DNA (cDNA) synthesis, and inhibit RT–PCR amplification [8–11]. To this point, a well-defined method to extract RNA from biofilms has not been published; most of the currently used RNA isolation protocols for microbial biofilms are derived from those used in planktonic cells, which are not
Extraction of total RNA from S. mutans biofilms / J.A. Cury, H. Koo / Anal. Biochem. 365 (2007) 208–214
optimized in terms of yield and/or genomic DNA removal [12]. Therefore, an RNA extraction and purification method that consistently yields high-quality RNA from biofilms in sufficient quantity for RNA-based experiments such as real-time PCR would facilitate the study of gene expression in biofilms. In this study, several methods of RNA extraction and purification were compared using Streptococcus mutans in biofilms as a source of RNA. This oral pathogen was selected because of its ability to form biofilms on the tooth surface by synthesizing complex extracellular polysaccharides from dietary sucrose and starch hydrolysates [13]. Materials and methods Preparation of biofilms of S. mutans UA159 Biofilms of S. mutans UA159 ATCC 700610 were formed on standard glass microscope slides (surface area 37.5 cm2, Micro Slides, VWR Scientific, West Chester, PA, USA) in batch cultures for 5 days as detailed elsewhere [14,15]. S. mutans UA159 is a well-described biofilm-forming and cariogenic dental pathogen and the strain selected for genomic sequencing [16]. Biofilm (5 days old, 84 mg dry weight or 1500 mg wet weight) was removed from the glass slides by means of a sterile spatula, split into three samples (28 mg dry weight each), and also kept in RNAlater solution. Biomass between 25 and 30 mg dry weight (of which 40% is extracellular polysaccharides) was the optimum amount for crude RNA extraction, according to our preliminary experiments and the protocol described in this study. Biofilm homogenization and polysaccharide removal The RNAlater solution was carefully removed using an automatic pipettor without disturbing the biofilm. The biofilm (28 mg dry weight) was either (i) resuspended with 7.0 ml of cold phosphate-buffered saline (PBS), vortexed, and centrifuged at 5500g, 4 °C, for 10 min, with the pellet being washed two more times using this procedure (three times total); or (ii) resuspended with 7.0 ml of cold PBS and homogenized by sonication using one 30-s pulse at outputs ranging from 5 to 9 W (Branson Ultrasonics, Danbury, CT, USA), with the suspension being centrifuged at 5500g, 4 °C, for 10 min and then the pellet being resuspended and sonicated again. This sonication–washing procedure was done one more time (three times total). All of the supernatants were retrieved, and the amount of polysaccharide removed from the biofilm’s matrix was determined as detailed elsewhere [14]. Briefly, the supernatants were pooled and 3 volumes of cold ethanol were added, and the resulting precipitate was collected. The precipitate, or water-soluble polysaccharide, was collected by centrifugation, washed with ice-cold 75% (v/v) ethanol three times followed by 99% ethanol, and dried in a SpeedVac concentrator. The pellet was resuspended in 50 ll of water, and
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the amount of carbohydrates was measured using the phenol-sulfuric method [17] with glucose as standard. After the washing (no sonication) or washing–sonication procedure, the biofilm suspension was centrifuged at 5500g, 4 °C, for 10 min and the pellets were subjected to RNA extraction. Extraction of total RNA The RNA from the biofilm pellets was extracted according to Chen and coworkers [3] and Kuhnert and Quivey [6] with some modifications. Briefly, the pellets (28 mg dry weight) were resuspended in 0.75 ml of NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and vortexed for 1 min using a vortexer at maximum speed. An equal volume of acid phenol/chloroform (5:1, pH 4.5, Ambion, Austin, TX, USA) was added to the suspension, vortexed for 30 s, and then transferred to a 2.0-ml screw-cap microcentrifuge tubes containing 0.8 g of glass beads (0.5 mm diameter, Biospec Products, Bartlesville, OK, USA). The cells were lysed in a Mini-Bead Beater homogenizer (Biospec Products) kept at 4 °C for a total of 120 s (beat three times for 40 s with 1-min interval). The homogenized suspension was centrifuged at 10,000g for 5 min at 4 °C, and the aqueous phase was collected and transferred to a microcentrifuge tube to which 0.75 ml of acid phenol/chloroform (5:1, pH 4.5) was added. Additional phenol/chloroform extractions (up to three times) were also examined. The mixture was vortexed for 10 s and centrifuged at 13,000g for 5 min at 4 °C. The aqueous phase was collected and extracted with a 1:1 solution of chloroform/isoamyl alcohol (24:1, Ambion). Alternatively, the aqueous phase was mixed with ethanol to a final concentration of 45% in the presence or absence of salt (sodium chloride or sodium acetate, up to 3 M) in an attempt to precipitate polysaccharides prior to RNA precipitation as described previously [18,19]. Total RNA was precipitated using a 1/10 volume of 3 M sodium acetate (pH 5.0) and 1 volume of isopropanol at 20 °C for at least 30 min. We also examined other commonly used RNA extraction procedures, including commercial kits using lysing matrix, enzymes (mutanolysin/lysozyme), and Trizol, but the RNA yield was 1.5 to 3.0 times lower than the mechanical cells disruption in NAES–acid phenol/chloroform used in this study. The RNA extraction protocol is shown in Fig. 1. DNase treatments The RNA precipitates were recovered by centrifugation (13,000g, 4 °C, 15 min), and the pellet was washed with icecold 75% (v/v) ethanol three times followed by 99% ethanol. The crude RNA was resuspended in molecular grade water and quantified spectrophotometrically (absorbance at 260 nm, A260). Extracted crude RNA was treated enzymatically with DNase I to remove contaminant genomic DNA. Two types of commercially available DNase I
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1) Remove the RNAlater above the biofilm sediment with an automatic pipettor; 2) Add cold 1× PBS, vortex, centrifuge (5500g at room temperature for 10 min). Discard the supernatant; 3) Resuspend pellet in 7 ml of cold 1× PBS (vortex), sonicate at 7 W for 30 s, and centrifuge (5500g at 4 oC for 10 min). Discard supernatant. Repeat this step two more times (three sonication-washings); 4) Add 0.75 ml of NAES buffer**. Resuspend pellet by vortex for 1 min; **50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS at pH 5.
5) Add 0.75 ml of acid phenol/chloroform (5:1, pH 4.5) and mix well (vortex); 6) Transfer the mixture to a 2-ml screw-cap tube containing 0.8 g of glass beads (0.5 mm diameter); 7) Bead beat for 40 s., and then let stand at 4 oC for 1 min. Repeat two more times; 8) Spin sample at 10,000g at 4 oC for 5 min, retrieve the aqueous solution, and transfer it to a new Eppendorf tube; 9) Add equal volume of acid phenol/chloroform (5:1, pH 4.5). Invert to mix and gently vortex, let sit for 1 min at room temperature. Then spin sample at 14,000g at 4 oC for 5 min; 10) Repeat 9th step (additional acid phenol/chloroform extraction); 11) Transfer the aqueous phase to a new tube and add 0.75 ml (or equal volume) of chloroform/isoamyl alcohol (24:1). Invert to mix, gently vortex, and spin sample at 14,000g at 4 oC for 5 min; 12) Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate (pH 5). Mix and add equal final volume of ice-cold isopropanol, and keep at -20 oC for at least 30 min; 13) Recover RNA by centrifugation (14,000g at 4 oC for 15 min). Wash RNA pellet with 0.5 ml ice-cold 75% ETOH (three times) followed by 0.5 ml of ice-cold 99% ethanol; 14) Resuspend the pellet in 50 μl of molecular grade water, and quantify RNA at 260 nm (OD260); 15)
The crude RNA is ready for DNase treatments according to modified Qiagen RNeasy Mini Prep protocol* followed by Ambion Turbo DNase and RNA cleanup using Qiagen RNeasy MinElute protocol. *Additional column washing with RLT buffer containing 36% ethanol prior to RW1 buffer washing step (as described in RNeasy Mini Prep protocol).
Fig. 1. Protocol for RNA extraction from S. mutans biofilms.
treatments were used: (i) on-column DNase I (Qiagen RNeasy Mini Kit, Qiagen Sciences, Germantown, MD, USA) and (ii) protein-engineered DNase I in solution (Turbo DNase, Ambion). An aliquot of the extracted RNA ( 50 lg) was treated first using Qiagen’s RNeasy on-column DNase I (Q, 2.7 U DNase I/10 lg RNA) and then with Ambion’s Turbo DNase I (T, 2 U Turbo DNase I/10 lg RNA), followed by Qiagen RNeasy MinElute (for DNase I removal) according to the manufacturer’s protocols with the exception of Qiagen’s on-column DNase I. The RNeasy on-column DNase I protocol was modified by adding a washing step with 350 ll of RLT buffer containing 36% ethanol prior to column washing with RW1 buffer (RLT and RW1 buffers are proprietary solutions of Qiagen Sciences). The additional washing improved the polysaccharide removal and RNA recovery. The combination of DNase I treatments (Q followed by T) provided
better genomic DNA removal than did either treatment alone or other combinations (J. A. Cury et al., unpublished data). Determination of RNA purity and integrity The quality of the purified RNA was examined by (i) an Agilent 2100 electrophoresis bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) [20] and (ii) the absorbance ratio A260/A280 in Tris buffer (10 mM, pH 7.5) [21]. Carbohydrate analysis of RNA extracts The total concentration of carbohydrates in crude preparations of RNA was quantified by the phenol-sulfuric method using glucose as standard [17]. Nucleic acids react in this method [8]; therefore, a correction was applied using
Extraction of total RNA from S. mutans biofilms / J.A. Cury, H. Koo / Anal. Biochem. 365 (2007) 208–214
Real-time RT–PCR analysis To check for genomic DNA contamination, 1 lg of purified RNA (without RT) from each of the DNase treatment procedures was amplified by a MyiQ real-time PCR detection system with iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) containing specific primer sets for gtfB [5,22]. In addition, cDNAs were synthesized from 1 lg of purified RNA samples using a Bio-Rad iScript cDNA synthesis kit, which contains an MMLV RNase H+RT and random hexamers. The resulting cDNA was used as template in the real-time PCR step, and a reaction containing only the reagents (no template control) was also included. The critical threshold cycle (Ct) was defined as the cycle at which the fluorescence becomes detectable above the background and is inversely proportional to the logarithm of the initial number of template molecules. Statistical analysis Triplicates were conducted in each of the experiments. The data were analyzed using analysis of variance (ANOVA), and the F test was used to test any differences between the groups. When significant differences were detected, pairwise comparisons were made among all of the groups using Tukey’s method to adjust for multiple comparisons. Statistical software JMP (version 3.1, SAS Institute, Cary, NC, USA) was used to perform the analyses. The level of significance was set at 5%. Results and discussion The current procedures for RNA extraction and removal of contaminant substances from microorganisms enmeshed in a polysaccharide-rich biofilms matrix are unsatisfactory [12]; most of them are adapted from RNA isolation procedures used for planktonic cells. Polysaccharides interfere with RNA extraction, cDNA synthesis, RT– PCR, and hybridization in Northern analyses [8,11,23] and, therefore, should be eliminated during the RNA purification. The current study examined several protocols for RNA extraction and extracellular polysaccharide removal from S. mutans biofilms, resulting in a method that yields high-quality RNA with negligible amounts of polysaccharides and contaminant genomic DNA (see detailed RNA extraction method in Fig. 1). First, we examined the influence of sonication on extracellular polysaccharide removal from the biofilms. The ultrasonics principle involves the generation of shock waves from implosion of micro bubbles that could homogenize the biofilms by dispersing the microbial cells and polysaccharides from the extracellular matrix; this procedure is not necessary for RNA isolation in planktonic cells.
Therefore, the biofilms were subjected to the washing-only (with no sonication, termed protocol 1) or sonication– washing (termed protocol 2) procedures. The best polysaccharide removal was achieved by three sonication–washing steps using a 30-s pulse at 7 W output (protocol 2), and the results are shown in Fig. 2. Clearly, the sonication of the biofilms is a critical step to remove water-soluble polysaccharides from biofilm’s matrix; biofilms without sonication (protocol 1) showed minimal polysaccharide removal. The yield and purity of the crude preparations of RNA extracted from biofilms of S. mutans were also affected due to biofilm homogenization by sonication, as shown in Table 1. The biofilms subjected to the sonication–washing procedure showed 3.2 times more RNA and 3.9 times less polysaccharide in the RNA extract than did those without sonication. The A260/A280 ratios were 2.1 irrespective of whether the biofilms were subjected to the sonication– washing procedure, an observation that suggests little or no protein contamination. In contrast, the polysaccharide ratio, [carbohydrate] / [RNA], of the RNA extract from sonicated biofilms was 2.2 times lower than that without biofilm homogenization (0.33 vs. 0.74). Nevertheless, the 0.33 ratio of the RNA extracts obtained using protocol 2 still indicates the presence of contaminant polysaccharides given that the ratio for pure RNA (Sigma) is 0.19. Therefore, we attempted to improve the polysaccharide removal during the RNA extraction step. The polysaccharides from S. mutans biofilms’ matrix are composed mostly of insoluble a1,3-linked glucan and soluble a1,6-linked glucan [13,14,24]. We examined the solubility of insoluble glucans (synthesized by purified glucosyltransferase B) and soluble glucans (synthesized by purified glucosyltransferase D) in the mixture of NAES buffer/phenol/chloroform in the presence of glass beads (after bead beading) to simulate the exact conditions of the RNA extraction procedure. The aqueous phase contained mostly soluble glucans, whereas insoluble glucans were found in the phenol/chloroform phase. Thus, additional phenol/chloroform extractions and ethanol/salt precipitation of the aqueous phase 2.50
Milligrams of polysaccharide removed/biofilm
pure RNA (R6750, Sigma–Aldrich, St. Louis, MO, USA) as standard. In addition, the total carbohydrate concentration/RNA concentration ratio was determined.
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2.00 1.50 1.00 0.50 0.00 No sonication
With sonication
Fig. 2. Means and standard deviations (n = 3) of polysaccharide removed from biofilm matrix (mg/biofilm) by the washing-only (protocol 1) and sonication–washing (protocol 2) procedures. Values are significantly different from each other (P < 0.05, ANOVA, comparison for all pairs using Tukey’s test).
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Table 1 Yield and purity of RNA extracts from S. mutans biofilm according to extraction protocols tested before DNase treatments
A260/A280
*
*
*
*
Values (means ± SD, n = 3) marked with an asterisk are not significantly different from each other (P > 0.05, ANOVA, comparison for all pairs using Tukey’s test). The yield in planktonic cells using these protocols is between 2 and 4 mg RNA/100 mg of cell dry weight. a RNA standard (Sigma) = 2.10. b RNA standard (Sigma) = 0.19. c Protocol 1: washed three times with PBS. d Protocol 2: sonication–washing (three times). e Protocol 3: sonication–washing (three times) + 1 additional phenol/chloroform treatment. f Protocol 4: sonication–washing (three times) + 45% ethanol precipitation.
were tested to improve the polysaccharide removal from the RNA extracts. An additional phenol/chloroform extraction (protocol 3) improved the polysaccharide removal without significantly compromising the RNA yield, as shown in Table 1; more extractions affected the RNA yield and did not further increase the polysaccharide removal. Ethanol precipitation (25 45% with or without salts, protocol 4) did not enhance the polysaccharide removal, and the RNA yield was adversely affected, probably due to coprecipitation of RNA. It is apparent that most of the soluble polysaccharides are removed during the sonication procedure, whereas the insoluble polysaccharides are extracted during phenol/chloroform extraction alongside bacterial membrane and debris. Therefore, RNA extracts using protocol 3 were selected for further purification and analyses. For successful isolation of intact RNA, it is also important to remove as much contaminant genomic DNA as possible in an RNA preparation [1,25]. The removal of genomic DNA is also particularly difficult in biofilms [12], possibly due to the presence of an extracellular polysaccharide matrix. A combination of Qiagen’s RNeasy on-column DNase I digestion followed by Ambion’s Turbo DNase I
was shown to be the most effective approach for genomic DNA removal in S. mutans (J. A. Cury et al., unpublished data). The yield after DNase I treatments was 92.0 ± 0.6 lg of RNA/100 mg of biofilm dry weight, as shown in Table 2; for comparison, the RNA yield using standard protocols (without sonication) is approximately 20 lg of RNA/100 mg of biofilm dry weight. The [carbohydrate]/ [RNA] ratio for the RNA from biofilms was 0.23, indicating that most, if not all, of the contaminant polysaccharide was removed; all of the A260/A280 ratios were above 2.18. In addition, the integrity of RNA was examined by labon-chip capillary electrophoresis (Agilent Bioanalyzer 2100) [20], as shown in Fig. 3. The microfluidic capillary electrophoresis showed sharp and distinct 23 S and 16 S ribosomal RNA bands with minimal degradation. The Bioanalyzer 2100 also provides the RNA integrity number (RIN), a new tool for RNA quality assessment [1]. This tool is based on a neuronal network that determines the RIN from the shape of the curve in the electropherogram (Fig. 3). The software and algorithm allow the classification of total RNA on a numbering system from 1 (most degraded profile) to 10 (most intact profile). According to Fleige and Pfaffl [1], a RIN greater than 5 is considered
Table 2 Yield, purity, and real-time RT–PCR analysis of RNA extract after DNase treatmentsa Protocol
Yield (lg RNA/100 mg biofilm dry weight)
RNA analyses Ratio b
3
92.0 ± 0.6
A260/A280 nm
[Carbohydrate] / [RNA]c
2.18 ± 0.007
0.23 ± 0.02
Polysaccharide (lg/ml of RNA extract)
gtfB (Ct)d
1.8 ± 1.2
33.6 ± 0.7
Values are means ± SD (n = 3). RNA purified from crude extracts obtained using protocol 1 (no sonication) showed Ct values as early as 26 cycles. a RNeasy protocol + additional washing with RLT + Turbo DNase treatment + RNeasy cleanup. b RNA standard (Sigma) = 2.10. c RNA standard (Sigma) = 0.19. d Purified RNA was used as template in the presence of SYBR Green I and gtfB primer sets.
Extraction of total RNA from S. mutans biofilms / J.A. Cury, H. Koo / Anal. Biochem. 365 (2007) 208–214
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Fig. 3. Analysis of RNA integrity of purified sample using protocol 3 by lab-on-chip capillary electrophoresis.
to be a good total RNA quality for downstream applications such as qRT–PCR. In the current study, the RIN of the purified RNA sample was 9.0. The quality of RNA preparations was also analyzed using real-time RT–PCR, SYBR Green I, and primer sets for gtfB, which was selected because it is a critical gene associated with extracellular polysaccharide synthesis [26] and is commonly used for S. mutans detection in clinical samples using qRT–PCR-based experiments [27]. First, the purified RNA samples (1 lg) were used as templates in PCR reaction to examine for the presence of residual genomic DNA by monitoring the increasing fluorescence intensity after each PCR cycle; SYBR Green I fluorescence dye binds specifically to the minor groove double-stranded DNA [28]. A value, Ct, is calculated based on the time (measured in PCR cycle numbers) at which the fluorescence emission increases beyond a threshold level (based on the background fluorescence of the system); a greater amount of DNA results in a lower Ct value as a result of requiring fewer PCR cycles for the fluorescence emission intensity to reach the threshold. The RNA extracts using the method outlined in this study showed detectable fluorescence signal only after 33.6 cycles when using the primer sets for gtfB, indicating negligible amounts of DNA. For comparison, RNA purified from crude extracts obtained using protocol 1 (no sonication) showed fluorescence signal as early as 26 cycles, indicating significant amounts of genomic DNA (data not shown). Finally, cDNA pools were synthesized from 1 lg of purified RNA using random hexamers. The amplification (with gtfB primers) of either two- or fivefold serial dilution
of cDNAs from RNA purified according to the selected method provided correlation coefficients of 0.98 to 0.99 and slopes between 3.289 and 3.365 (98.2 101.4% amplification efficiency), within the range of acceptable slope ( 3.0 to 3.5), indicating little or no PCR inhibitors. The cDNAs diluted as much as 50 times were successfully amplified, showing Ct values between 26.5 and 27.5. The no-template control showed negligible amplification (>34 cycles). In conclusion, a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for realtime RT–PCR analyses has been described in this study. Although we focused on a single albeit important organism, we believe that our results will have importance for additional organisms present in biofilms. Acknowledgments The authors are grateful to William Bowen for critical reading of the manuscript prior to submission and to Simone Duarte and Jennifer Seils for technical assistance. This research was supported in part by the U.S. Department of Agriculture (USDA, research grant 2006-3520016589) and the Brazilian Funding Agency–Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES, research grant BEX 0494/05-4). References [1] S. Fleige, M.W. Pfaffl, RNA integrity and the effect on the real-time qRT–PCR performance, Mol. Aspects Med. 27 (2006) 126–139.
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Extraction and purification of total RNA from Sreptococcus mutans biofilms Jaime A. Cury a, Hyun Koo b
b,*
a Faculty of Dentistry of Piracicaba, State University of Campinas, Piracicaba, Sa˜o Paulo, Brazil Eastman Department of Dentistry and Center for Oral Biology, University of Rochester Medical Center, Rochester, NY 14620, USA
Received 29 January 2007 Available online 24 March 2007
Abstract RNA isolation from Streptococcus mutans within biofilms is challenging because of the presence of extracellular polysaccharide matrix that interferes with RNA extraction procedures. In an effort to solve this difficult problem, we examined several protocols to extract and purify RNA from S. mutans biofilms. A combination of sonication (three times using a 30-s pulse at 7 W) with washing in phosphate-buffered saline removed most of the extracellular polysaccharides from the biofilms and provided the highest RNA yield. Further homogenization–mechanical cells disruption in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and acid phenol/chloroform yielded 547.2 ± 23.4 lg RNA/100 mg of biofilm dry weight. An additional acid phenol/chloroform extraction further improved the purification of RNA without significantly affecting the RNA yield. The combination of DNase I in silica gel-based column and recombinant DNase I in solution effectively removed the genomic DNA as determined by real-time quantitative reverse transcriptase PCR (RT–PCR), resulting in 92.0 ± 0.6 lg of purified RNA per 100 mg of biofilm dry weight. The complementary DNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results demonstrated a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for real-time RT–PCR analyses, and our data have relevance for isolation of RNA from other biofilm-forming microorganisms. Ó 2007 Elsevier Inc. All rights reserved. Keywords: RNA extraction; Real-time PCR; DNase I; Streptococcus mutans; Biofilms
Isolation of high-quality intact RNA with minimum levels of contaminants and in sufficient quantity is critical for downstream applications using functional genomics approaches, especially real-time quantitative reverse transcriptase PCR (qRT PCR)1 [1]. Real-time RT–PCR is a sensitive and reliable technique to measure gene expression but requires a reproducible and well-defined methodology for RNA extraction and purification for accurate determination of messenger RNA (mRNA) levels.
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Corresponding author. Fax: +1 585 276 0190. E-mail address: [email protected] (H. Koo). 1 Abbreviations used: qRT–PCR, quantitative reverse transcriptase PCR; mRNA, messenger RNA; cDNA, complementary DNA; PBS, phosphatebuffered saline; ANOVA, analysis of variance; RIN, RNA integrity number. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.03.021
Several methods of RNA extraction and purification have been reported for bacteria grown in a planktonic state, including oral streptococci [2–7]. In contrast to planktonic cells, RNA isolation from microorganisms within biofilms is challenging because of the presence of an extracellular polysaccharide matrix that may interfere with the extraction and purification of the nucleic acids. RNA isolation and purification from polysaccharide-rich tissues, such as in plants and fruits, are difficult because these polymeric substances bind to nucleic acids, interfere with DNase activity, inhibit complementary DNA (cDNA) synthesis, and inhibit RT–PCR amplification [8–11]. To this point, a well-defined method to extract RNA from biofilms has not been published; most of the currently used RNA isolation protocols for microbial biofilms are derived from those used in planktonic cells, which are not
Extraction of total RNA from S. mutans biofilms / J.A. Cury, H. Koo / Anal. Biochem. 365 (2007) 208–214
optimized in terms of yield and/or genomic DNA removal [12]. Therefore, an RNA extraction and purification method that consistently yields high-quality RNA from biofilms in sufficient quantity for RNA-based experiments such as real-time PCR would facilitate the study of gene expression in biofilms. In this study, several methods of RNA extraction and purification were compared using Streptococcus mutans in biofilms as a source of RNA. This oral pathogen was selected because of its ability to form biofilms on the tooth surface by synthesizing complex extracellular polysaccharides from dietary sucrose and starch hydrolysates [13]. Materials and methods Preparation of biofilms of S. mutans UA159 Biofilms of S. mutans UA159 ATCC 700610 were formed on standard glass microscope slides (surface area 37.5 cm2, Micro Slides, VWR Scientific, West Chester, PA, USA) in batch cultures for 5 days as detailed elsewhere [14,15]. S. mutans UA159 is a well-described biofilm-forming and cariogenic dental pathogen and the strain selected for genomic sequencing [16]. Biofilm (5 days old, 84 mg dry weight or 1500 mg wet weight) was removed from the glass slides by means of a sterile spatula, split into three samples (28 mg dry weight each), and also kept in RNAlater solution. Biomass between 25 and 30 mg dry weight (of which 40% is extracellular polysaccharides) was the optimum amount for crude RNA extraction, according to our preliminary experiments and the protocol described in this study. Biofilm homogenization and polysaccharide removal The RNAlater solution was carefully removed using an automatic pipettor without disturbing the biofilm. The biofilm (28 mg dry weight) was either (i) resuspended with 7.0 ml of cold phosphate-buffered saline (PBS), vortexed, and centrifuged at 5500g, 4 °C, for 10 min, with the pellet being washed two more times using this procedure (three times total); or (ii) resuspended with 7.0 ml of cold PBS and homogenized by sonication using one 30-s pulse at outputs ranging from 5 to 9 W (Branson Ultrasonics, Danbury, CT, USA), with the suspension being centrifuged at 5500g, 4 °C, for 10 min and then the pellet being resuspended and sonicated again. This sonication–washing procedure was done one more time (three times total). All of the supernatants were retrieved, and the amount of polysaccharide removed from the biofilm’s matrix was determined as detailed elsewhere [14]. Briefly, the supernatants were pooled and 3 volumes of cold ethanol were added, and the resulting precipitate was collected. The precipitate, or water-soluble polysaccharide, was collected by centrifugation, washed with ice-cold 75% (v/v) ethanol three times followed by 99% ethanol, and dried in a SpeedVac concentrator. The pellet was resuspended in 50 ll of water, and
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the amount of carbohydrates was measured using the phenol-sulfuric method [17] with glucose as standard. After the washing (no sonication) or washing–sonication procedure, the biofilm suspension was centrifuged at 5500g, 4 °C, for 10 min and the pellets were subjected to RNA extraction. Extraction of total RNA The RNA from the biofilm pellets was extracted according to Chen and coworkers [3] and Kuhnert and Quivey [6] with some modifications. Briefly, the pellets (28 mg dry weight) were resuspended in 0.75 ml of NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA, and 1% SDS, pH 5.0) and vortexed for 1 min using a vortexer at maximum speed. An equal volume of acid phenol/chloroform (5:1, pH 4.5, Ambion, Austin, TX, USA) was added to the suspension, vortexed for 30 s, and then transferred to a 2.0-ml screw-cap microcentrifuge tubes containing 0.8 g of glass beads (0.5 mm diameter, Biospec Products, Bartlesville, OK, USA). The cells were lysed in a Mini-Bead Beater homogenizer (Biospec Products) kept at 4 °C for a total of 120 s (beat three times for 40 s with 1-min interval). The homogenized suspension was centrifuged at 10,000g for 5 min at 4 °C, and the aqueous phase was collected and transferred to a microcentrifuge tube to which 0.75 ml of acid phenol/chloroform (5:1, pH 4.5) was added. Additional phenol/chloroform extractions (up to three times) were also examined. The mixture was vortexed for 10 s and centrifuged at 13,000g for 5 min at 4 °C. The aqueous phase was collected and extracted with a 1:1 solution of chloroform/isoamyl alcohol (24:1, Ambion). Alternatively, the aqueous phase was mixed with ethanol to a final concentration of 45% in the presence or absence of salt (sodium chloride or sodium acetate, up to 3 M) in an attempt to precipitate polysaccharides prior to RNA precipitation as described previously [18,19]. Total RNA was precipitated using a 1/10 volume of 3 M sodium acetate (pH 5.0) and 1 volume of isopropanol at 20 °C for at least 30 min. We also examined other commonly used RNA extraction procedures, including commercial kits using lysing matrix, enzymes (mutanolysin/lysozyme), and Trizol, but the RNA yield was 1.5 to 3.0 times lower than the mechanical cells disruption in NAES–acid phenol/chloroform used in this study. The RNA extraction protocol is shown in Fig. 1. DNase treatments The RNA precipitates were recovered by centrifugation (13,000g, 4 °C, 15 min), and the pellet was washed with icecold 75% (v/v) ethanol three times followed by 99% ethanol. The crude RNA was resuspended in molecular grade water and quantified spectrophotometrically (absorbance at 260 nm, A260). Extracted crude RNA was treated enzymatically with DNase I to remove contaminant genomic DNA. Two types of commercially available DNase I
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1) Remove the RNAlater above the biofilm sediment with an automatic pipettor; 2) Add cold 1× PBS, vortex, centrifuge (5500g at room temperature for 10 min). Discard the supernatant; 3) Resuspend pellet in 7 ml of cold 1× PBS (vortex), sonicate at 7 W for 30 s, and centrifuge (5500g at 4 oC for 10 min). Discard supernatant. Repeat this step two more times (three sonication-washings); 4) Add 0.75 ml of NAES buffer**. Resuspend pellet by vortex for 1 min; **50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS at pH 5.
5) Add 0.75 ml of acid phenol/chloroform (5:1, pH 4.5) and mix well (vortex); 6) Transfer the mixture to a 2-ml screw-cap tube containing 0.8 g of glass beads (0.5 mm diameter); 7) Bead beat for 40 s., and then let stand at 4 oC for 1 min. Repeat two more times; 8) Spin sample at 10,000g at 4 oC for 5 min, retrieve the aqueous solution, and transfer it to a new Eppendorf tube; 9) Add equal volume of acid phenol/chloroform (5:1, pH 4.5). Invert to mix and gently vortex, let sit for 1 min at room temperature. Then spin sample at 14,000g at 4 oC for 5 min; 10) Repeat 9th step (additional acid phenol/chloroform extraction); 11) Transfer the aqueous phase to a new tube and add 0.75 ml (or equal volume) of chloroform/isoamyl alcohol (24:1). Invert to mix, gently vortex, and spin sample at 14,000g at 4 oC for 5 min; 12) Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate (pH 5). Mix and add equal final volume of ice-cold isopropanol, and keep at -20 oC for at least 30 min; 13) Recover RNA by centrifugation (14,000g at 4 oC for 15 min). Wash RNA pellet with 0.5 ml ice-cold 75% ETOH (three times) followed by 0.5 ml of ice-cold 99% ethanol; 14) Resuspend the pellet in 50 μl of molecular grade water, and quantify RNA at 260 nm (OD260); 15)
The crude RNA is ready for DNase treatments according to modified Qiagen RNeasy Mini Prep protocol* followed by Ambion Turbo DNase and RNA cleanup using Qiagen RNeasy MinElute protocol. *Additional column washing with RLT buffer containing 36% ethanol prior to RW1 buffer washing step (as described in RNeasy Mini Prep protocol).
Fig. 1. Protocol for RNA extraction from S. mutans biofilms.
treatments were used: (i) on-column DNase I (Qiagen RNeasy Mini Kit, Qiagen Sciences, Germantown, MD, USA) and (ii) protein-engineered DNase I in solution (Turbo DNase, Ambion). An aliquot of the extracted RNA ( 50 lg) was treated first using Qiagen’s RNeasy on-column DNase I (Q, 2.7 U DNase I/10 lg RNA) and then with Ambion’s Turbo DNase I (T, 2 U Turbo DNase I/10 lg RNA), followed by Qiagen RNeasy MinElute (for DNase I removal) according to the manufacturer’s protocols with the exception of Qiagen’s on-column DNase I. The RNeasy on-column DNase I protocol was modified by adding a washing step with 350 ll of RLT buffer containing 36% ethanol prior to column washing with RW1 buffer (RLT and RW1 buffers are proprietary solutions of Qiagen Sciences). The additional washing improved the polysaccharide removal and RNA recovery. The combination of DNase I treatments (Q followed by T) provided
better genomic DNA removal than did either treatment alone or other combinations (J. A. Cury et al., unpublished data). Determination of RNA purity and integrity The quality of the purified RNA was examined by (i) an Agilent 2100 electrophoresis bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) [20] and (ii) the absorbance ratio A260/A280 in Tris buffer (10 mM, pH 7.5) [21]. Carbohydrate analysis of RNA extracts The total concentration of carbohydrates in crude preparations of RNA was quantified by the phenol-sulfuric method using glucose as standard [17]. Nucleic acids react in this method [8]; therefore, a correction was applied using
Extraction of total RNA from S. mutans biofilms / J.A. Cury, H. Koo / Anal. Biochem. 365 (2007) 208–214
Real-time RT–PCR analysis To check for genomic DNA contamination, 1 lg of purified RNA (without RT) from each of the DNase treatment procedures was amplified by a MyiQ real-time PCR detection system with iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) containing specific primer sets for gtfB [5,22]. In addition, cDNAs were synthesized from 1 lg of purified RNA samples using a Bio-Rad iScript cDNA synthesis kit, which contains an MMLV RNase H+RT and random hexamers. The resulting cDNA was used as template in the real-time PCR step, and a reaction containing only the reagents (no template control) was also included. The critical threshold cycle (Ct) was defined as the cycle at which the fluorescence becomes detectable above the background and is inversely proportional to the logarithm of the initial number of template molecules. Statistical analysis Triplicates were conducted in each of the experiments. The data were analyzed using analysis of variance (ANOVA), and the F test was used to test any differences between the groups. When significant differences were detected, pairwise comparisons were made among all of the groups using Tukey’s method to adjust for multiple comparisons. Statistical software JMP (version 3.1, SAS Institute, Cary, NC, USA) was used to perform the analyses. The level of significance was set at 5%. Results and discussion The current procedures for RNA extraction and removal of contaminant substances from microorganisms enmeshed in a polysaccharide-rich biofilms matrix are unsatisfactory [12]; most of them are adapted from RNA isolation procedures used for planktonic cells. Polysaccharides interfere with RNA extraction, cDNA synthesis, RT– PCR, and hybridization in Northern analyses [8,11,23] and, therefore, should be eliminated during the RNA purification. The current study examined several protocols for RNA extraction and extracellular polysaccharide removal from S. mutans biofilms, resulting in a method that yields high-quality RNA with negligible amounts of polysaccharides and contaminant genomic DNA (see detailed RNA extraction method in Fig. 1). First, we examined the influence of sonication on extracellular polysaccharide removal from the biofilms. The ultrasonics principle involves the generation of shock waves from implosion of micro bubbles that could homogenize the biofilms by dispersing the microbial cells and polysaccharides from the extracellular matrix; this procedure is not necessary for RNA isolation in planktonic cells.
Therefore, the biofilms were subjected to the washing-only (with no sonication, termed protocol 1) or sonication– washing (termed protocol 2) procedures. The best polysaccharide removal was achieved by three sonication–washing steps using a 30-s pulse at 7 W output (protocol 2), and the results are shown in Fig. 2. Clearly, the sonication of the biofilms is a critical step to remove water-soluble polysaccharides from biofilm’s matrix; biofilms without sonication (protocol 1) showed minimal polysaccharide removal. The yield and purity of the crude preparations of RNA extracted from biofilms of S. mutans were also affected due to biofilm homogenization by sonication, as shown in Table 1. The biofilms subjected to the sonication–washing procedure showed 3.2 times more RNA and 3.9 times less polysaccharide in the RNA extract than did those without sonication. The A260/A280 ratios were 2.1 irrespective of whether the biofilms were subjected to the sonication– washing procedure, an observation that suggests little or no protein contamination. In contrast, the polysaccharide ratio, [carbohydrate] / [RNA], of the RNA extract from sonicated biofilms was 2.2 times lower than that without biofilm homogenization (0.33 vs. 0.74). Nevertheless, the 0.33 ratio of the RNA extracts obtained using protocol 2 still indicates the presence of contaminant polysaccharides given that the ratio for pure RNA (Sigma) is 0.19. Therefore, we attempted to improve the polysaccharide removal during the RNA extraction step. The polysaccharides from S. mutans biofilms’ matrix are composed mostly of insoluble a1,3-linked glucan and soluble a1,6-linked glucan [13,14,24]. We examined the solubility of insoluble glucans (synthesized by purified glucosyltransferase B) and soluble glucans (synthesized by purified glucosyltransferase D) in the mixture of NAES buffer/phenol/chloroform in the presence of glass beads (after bead beading) to simulate the exact conditions of the RNA extraction procedure. The aqueous phase contained mostly soluble glucans, whereas insoluble glucans were found in the phenol/chloroform phase. Thus, additional phenol/chloroform extractions and ethanol/salt precipitation of the aqueous phase 2.50
Milligrams of polysaccharide removed/biofilm
pure RNA (R6750, Sigma–Aldrich, St. Louis, MO, USA) as standard. In addition, the total carbohydrate concentration/RNA concentration ratio was determined.
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2.00 1.50 1.00 0.50 0.00 No sonication
With sonication
Fig. 2. Means and standard deviations (n = 3) of polysaccharide removed from biofilm matrix (mg/biofilm) by the washing-only (protocol 1) and sonication–washing (protocol 2) procedures. Values are significantly different from each other (P < 0.05, ANOVA, comparison for all pairs using Tukey’s test).
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Table 1 Yield and purity of RNA extracts from S. mutans biofilm according to extraction protocols tested before DNase treatments
A260/A280
*
*
*
*
Values (means ± SD, n = 3) marked with an asterisk are not significantly different from each other (P > 0.05, ANOVA, comparison for all pairs using Tukey’s test). The yield in planktonic cells using these protocols is between 2 and 4 mg RNA/100 mg of cell dry weight. a RNA standard (Sigma) = 2.10. b RNA standard (Sigma) = 0.19. c Protocol 1: washed three times with PBS. d Protocol 2: sonication–washing (three times). e Protocol 3: sonication–washing (three times) + 1 additional phenol/chloroform treatment. f Protocol 4: sonication–washing (three times) + 45% ethanol precipitation.
were tested to improve the polysaccharide removal from the RNA extracts. An additional phenol/chloroform extraction (protocol 3) improved the polysaccharide removal without significantly compromising the RNA yield, as shown in Table 1; more extractions affected the RNA yield and did not further increase the polysaccharide removal. Ethanol precipitation (25 45% with or without salts, protocol 4) did not enhance the polysaccharide removal, and the RNA yield was adversely affected, probably due to coprecipitation of RNA. It is apparent that most of the soluble polysaccharides are removed during the sonication procedure, whereas the insoluble polysaccharides are extracted during phenol/chloroform extraction alongside bacterial membrane and debris. Therefore, RNA extracts using protocol 3 were selected for further purification and analyses. For successful isolation of intact RNA, it is also important to remove as much contaminant genomic DNA as possible in an RNA preparation [1,25]. The removal of genomic DNA is also particularly difficult in biofilms [12], possibly due to the presence of an extracellular polysaccharide matrix. A combination of Qiagen’s RNeasy on-column DNase I digestion followed by Ambion’s Turbo DNase I
was shown to be the most effective approach for genomic DNA removal in S. mutans (J. A. Cury et al., unpublished data). The yield after DNase I treatments was 92.0 ± 0.6 lg of RNA/100 mg of biofilm dry weight, as shown in Table 2; for comparison, the RNA yield using standard protocols (without sonication) is approximately 20 lg of RNA/100 mg of biofilm dry weight. The [carbohydrate]/ [RNA] ratio for the RNA from biofilms was 0.23, indicating that most, if not all, of the contaminant polysaccharide was removed; all of the A260/A280 ratios were above 2.18. In addition, the integrity of RNA was examined by labon-chip capillary electrophoresis (Agilent Bioanalyzer 2100) [20], as shown in Fig. 3. The microfluidic capillary electrophoresis showed sharp and distinct 23 S and 16 S ribosomal RNA bands with minimal degradation. The Bioanalyzer 2100 also provides the RNA integrity number (RIN), a new tool for RNA quality assessment [1]. This tool is based on a neuronal network that determines the RIN from the shape of the curve in the electropherogram (Fig. 3). The software and algorithm allow the classification of total RNA on a numbering system from 1 (most degraded profile) to 10 (most intact profile). According to Fleige and Pfaffl [1], a RIN greater than 5 is considered
Table 2 Yield, purity, and real-time RT–PCR analysis of RNA extract after DNase treatmentsa Protocol
Yield (lg RNA/100 mg biofilm dry weight)
RNA analyses Ratio b
3
92.0 ± 0.6
A260/A280 nm
[Carbohydrate] / [RNA]c
2.18 ± 0.007
0.23 ± 0.02
Polysaccharide (lg/ml of RNA extract)
gtfB (Ct)d
1.8 ± 1.2
33.6 ± 0.7
Values are means ± SD (n = 3). RNA purified from crude extracts obtained using protocol 1 (no sonication) showed Ct values as early as 26 cycles. a RNeasy protocol + additional washing with RLT + Turbo DNase treatment + RNeasy cleanup. b RNA standard (Sigma) = 2.10. c RNA standard (Sigma) = 0.19. d Purified RNA was used as template in the presence of SYBR Green I and gtfB primer sets.
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Fig. 3. Analysis of RNA integrity of purified sample using protocol 3 by lab-on-chip capillary electrophoresis.
to be a good total RNA quality for downstream applications such as qRT–PCR. In the current study, the RIN of the purified RNA sample was 9.0. The quality of RNA preparations was also analyzed using real-time RT–PCR, SYBR Green I, and primer sets for gtfB, which was selected because it is a critical gene associated with extracellular polysaccharide synthesis [26] and is commonly used for S. mutans detection in clinical samples using qRT–PCR-based experiments [27]. First, the purified RNA samples (1 lg) were used as templates in PCR reaction to examine for the presence of residual genomic DNA by monitoring the increasing fluorescence intensity after each PCR cycle; SYBR Green I fluorescence dye binds specifically to the minor groove double-stranded DNA [28]. A value, Ct, is calculated based on the time (measured in PCR cycle numbers) at which the fluorescence emission increases beyond a threshold level (based on the background fluorescence of the system); a greater amount of DNA results in a lower Ct value as a result of requiring fewer PCR cycles for the fluorescence emission intensity to reach the threshold. The RNA extracts using the method outlined in this study showed detectable fluorescence signal only after 33.6 cycles when using the primer sets for gtfB, indicating negligible amounts of DNA. For comparison, RNA purified from crude extracts obtained using protocol 1 (no sonication) showed fluorescence signal as early as 26 cycles, indicating significant amounts of genomic DNA (data not shown). Finally, cDNA pools were synthesized from 1 lg of purified RNA using random hexamers. The amplification (with gtfB primers) of either two- or fivefold serial dilution
of cDNAs from RNA purified according to the selected method provided correlation coefficients of 0.98 to 0.99 and slopes between 3.289 and 3.365 (98.2 101.4% amplification efficiency), within the range of acceptable slope ( 3.0 to 3.5), indicating little or no PCR inhibitors. The cDNAs diluted as much as 50 times were successfully amplified, showing Ct values between 26.5 and 27.5. The no-template control showed negligible amplification (>34 cycles). In conclusion, a method that yields high-quality RNA from biofilms of S. mutans in sufficient quantity for realtime RT–PCR analyses has been described in this study. Although we focused on a single albeit important organism, we believe that our results will have importance for additional organisms present in biofilms. Acknowledgments The authors are grateful to William Bowen for critical reading of the manuscript prior to submission and to Simone Duarte and Jennifer Seils for technical assistance. This research was supported in part by the U.S. Department of Agriculture (USDA, research grant 2006-3520016589) and the Brazilian Funding Agency–Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES, research grant BEX 0494/05-4). References [1] S. Fleige, M.W. Pfaffl, RNA integrity and the effect on the real-time qRT–PCR performance, Mol. Aspects Med. 27 (2006) 126–139.
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