Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis
© França et al; licensee BioMed Central Ltd. 2011
Received: 20 October 2011
Accepted: 30 December 2011
Published: 30 December 2011
Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic resistance and tolerance to the immune system. Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Obtaining high quality mRNA from biofilms is crucial to validate the transcriptional measurements associated with the switching to the biofilm mode of growth. Therefore, we selected three commercially available RNA extraction kits with distinct characteristics, including those using silica membrane or organic extraction methods, and enzymatic or mechanical cell lysis, and evaluated the RNA quality obtained from two distinct S. epidermidis bacterial biofilms.
RNA extracted using the different kits was evaluated for quantity, purity, integrity, and functionally. All kits were able to extract intact and functional total RNA from the biofilms generated from each S. epidermidis strain. The results demonstrated that the kit based on mechanical lysis and organic extraction (FastRNA® Pro Blue) was the only one that was able to isolate pure and large quantities of RNA. Normalized expression of the icaA virulence gene showed that RNA extracted with PureLink™ had a significant lower concentration of icaA mRNA transcripts than the other kits tested.
When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Among the RNA extraction kits tested, FastRNA® Pro Blue was the best option for both S. epidermidis biofilms used.
Staphylococcus epidermidis, a normal inhabitant of normal human skin and mucosa, has recently emerged as a leading cause of biofilm-related infections, particularly, in patients with indwelling medical devices [1, 2] due to its ability to adhere to abiotic surfaces and to form biofilms [2, 3]. The quantification of specific messenger RNA (mRNA) from these biofilms is crucial to understanding the molecular mechanisms behind biofilm formation and maturation on the surface of medical devices. The success of any RNA-based analysis depends on the yield, purity, and integrity of the RNA [4, 5]. However, different RNA extraction methods can yield RNA with varying levels of quality [6, 7]. Currently, there are numerous methods for RNA extraction available, however there are only a few published studies comparing RNA extraction from biofilm samples [8–10]. Biofilm samples pose an increased problem to RNA extraction procedure mainly due to the presence of the extracellular matrix, which is estimated to comprise about 90% of the total biofilm biomass . Polysaccharides, the major component of the S. epidermidis biofilm matrix, seems to interfere with RNA extraction methods making bacterial cell lysis and nucleic acid purification difficult, and the purified RNA may still contain inhibitory substances [12, 13]. Therefore, in this study we compared three different commercially available RNA extraction kits to determine their ability to obtain high quantity, pure, intact, and functional RNA from S. epidermidis biofilms. The selected kits were based on distinct procedures and properties: FastRNA® Pro Blue (MPBiomedicals, Irvine, CA, US) uses mechanical and chemical lysis together with organic extraction; PureZOL™ RNA isolation reagent (Bio-Rad, Hercules, CA, US) uses chemical lysis with organic extraction, while PureLink™ RNA Mini Kit (Invitrogen, San Diego, CA, US) uses enzymatic lysis and silica membrane extraction.
Bacteria and growth conditions
S. epidermidis biofilms from two S. epidermidis strains with different genetic backgrounds (1457  and M187 ) were individually used to form separate biofilms. Strains were individually propagated by inoculating a single colony in 2 mL Tryptic Soy Broth (TSB) (Oxoid, Cambridge, UK) from plates not older than 2 days and grown at 37°C in a shaker rotator at 120 rpm for 24 (± 2) hours. Then, 10 μL of cell suspension was transferred to 2 mL of fresh TSB supplemented with 1% (w/v) of glucose to induce biofilm formation in a 24-well plate (Orange Scientific, Braine-L'Alleud, Belgium). The plate was incubated at 37°C with shaking at 100 rpm for 24 (± 2) hours. Prior to total RNA extraction, the culture media was removed and the biofilm was washed with 1 mL of NaCl 0.9% solution to remove planktonic cells. In order to count the total viable cells (CFUs/mL) within each S. epidermidis biofilm, the biofilms were resuspended in 1 mL of NaCl 0.9% solution and sonicated on ice for 10 s at 30 W. This procedure eliminates bacterial aggregates that do interfere with the CFUs counting . Subsequently, 10-fold dilutions in 0.9% NaCl were performed and plated on Tryptic Soy Agar (Oxoid) plates. The plates were then incubated at 37°C overnight.
Biofilm matrix composition
Biofilm total biomass, protein, and polysaccharide matrix content was determined as described previously . Briefly, the biofilm suspension was sonicated for 30 s at 30 W and, subsequently, centrifuged at 10.500 g for 6 min at 4°C. This procedure did not reduced cell viability as determined by CFU counting . The supernatants were then filtered through a 0.2 μm nitrocellulose filter and the proteins and polysaccharide content determined by Bicinchoninic Acid (BCA) protein assay  (G-Biosciences, MO, US) and Dubois method , respectively. The total biomass of the biofilms was determined by optical density at 595 nm. This experiment was performed in triplicates.
RNA extraction and quality indicators
Total RNA was isolated according the manufacturer's instructions, with the following modification, when appropriate: cell lysis was performed using 15 mg/mL of lysozyme for 60 min at 37°C with. This optimization increased the yield of total RNA 2-4-fold (data not shown). The final total RNA fraction was obtained by eluting or suspending the RNA in 45 μL of DEPC-treated water. To digest contaminating DNA, DNase (Fermentas, Burlington, Ontario, Canada) treatment was performed by adding 5 μL (10×) of reaction buffer with MgCl2 and 2 μL DNase I to the extracted RNA and incubating the mixture at 37°C for 30 min. Subsequently, 5 μL of 25 mM EDTA was added and incubated at 65°C for 10 min to inactivate the DNase I. Each experiment was performed in triplicates. The concentration and purity of the total RNA was spectrometrically assessed using a NanoDrop 1000™ (Thermo Scientific, Waltham, MA, US). The absorbance ratios A260/A280 were used as indicators of protein contamination and A260/A230 as indicators of polysaccharide, phenol, and/or chaotropic salts contamination . The integrity of the total RNA was assessed by visualization of the 23S/16S banding pattern. Electrophoresis was carried-out at 80 V for 60 min using a 1% agarose gel. The gel was stained with ethidium bromide and visualized using a GelDoc2000 (Bio-Rad). Total RNA extractions were performed two to four times.
Real time PCR (qPCR)
Primers used in cDNA synthesis and amplification by qPCR
Primers sequence (5' to 3')
Statistical significance of results was determined by unpaired t test using the Analysis Toolpak of Microsoft Excel 2007. P < 0.05 was considered to be statistically significant.
Results and Discussion
Comparison of the RNA yield and purity obtained by the three RNA extraction kits
RNA yield (ng/μl)
513 ± 135**
2.18 ± 0.06
2.06 ± 0.01**
359 ± 14**
2.09 ± 0.04
1.92 ± 0.06 **
18 ± 7
1.70 ± 0.06
0.30 ± 0.01
50 ± 7 *
1.70 ± 0.07
0.63 ± 0.16
17 ± 3
1.99 ± 0.08
1.35 ± 0.04
30 ± 3*
2.10 ± 0.06
1.25 ± 0.63
Besides the yield and purity, the extracted RNA should be intact and functional, in order to proper analyze the quantification of gene expression . RNA integrity was assessed by agarose gel electrophoresis as reported above, and the expected double banding pattern of the 23S/16S and the absence of a smear indicated the good integrity of RNA extracted (data not shown).
icaA expression values using RNA extracted from the three different kits
16S rRNA Ct
17.36 ± 0.41
31.48 ± 0.28
5.62 × 10-5
12.27 ± 0.81
27.07 ± 1.05
3.48 × 10-5
17.99 ± 0.30
31.84 ± 0.11
6.77 × 10-5
15.71 ± 0.83
29.58 ± 1.94
6.20 × 10-5
15.25 ± 0.12
32.56 ± 0.11
0.62 × 10-5 **
13.83 ± 0.67
31.13 ± 4.02
0.62 × 10-5 **
Observing the overall results it can be seen that depending on the RNA extraction kit used, the quantification of mRNA transcripts can be impaired. Interestingly, of all the parameters tested, RNA purity seemed to have a lower impact in inhibiting RNA quantification. The A260/280 and A260/230 ratios are just indicators of possible contaminants in the RNA sample. While some of the contaminants can interfere in the downstream applications, it seems that more than the concentration of contaminants, the nature of contaminants will impair RNA quantification. FastRNA® Pro Blue showed the best results, while PureLink™ RNA mini kit was the worst kit for S. epidermidis biofilm samples.
When working with complex samples, such as biofilms, that contain a high content extracellular polysaccharide and proteins, special care should be taken when selecting the appropriate RNA extraction system, in order to obtain accurate, reproducible, and biologically significant results. Testing different systems and probing for well described gene expression conditions might elucidate some less apparent pitfalls of RNA extraction kits.
List of Abbreviations
Colony-forming unit: Ct: cycle threshold
optical density: qPCR: Real time PCR
Tryptic Soy Broth.
This work was funded by European Union funds (FEDER/COMPETE) and by Portuguese national funds (FCT) under the projects with reference FCOMP-01-0124-FEDER-014309 (PTDC/BIA-MIC/113450/2009). AF and LDRM acknowledge the financial support of individual grants SFRH/BD/62359/2009 and SFRH/BD/66166/2009, respectively.
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