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Extraction and purification of total RNA from Sreptococcus mutans biofilms
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 b...
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| Published in: | Analytical biochemistry 2007-06, Vol.365 (2), p.208-214 |
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| Language: | English |
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| container_title | Analytical biochemistry |
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| creator | Cury, Jaime A. Koo, Hyun |
| description | 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
μg 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
μg 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. |
| doi_str_mv | 10.1016/j.ab.2007.03.021 |
| format | article |
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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
μg 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
μg 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.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/j.ab.2007.03.021</identifier><language>eng</language><publisher>Elsevier Inc</publisher><subject>Biofilms ; DNase I ; Real-time PCR ; RNA extraction ; Streptococcus mutans</subject><ispartof>Analytical biochemistry, 2007-06, Vol.365 (2), p.208-214</ispartof><rights>2007 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c325t-34170d799184cb8fb8e829bbcecef46e51f3dfc464d7d7ed9a6ca6c521421ad13</citedby><cites>FETCH-LOGICAL-c325t-34170d799184cb8fb8e829bbcecef46e51f3dfc464d7d7ed9a6ca6c521421ad13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,27957,27958</link.rule.ids></links><search><creatorcontrib>Cury, Jaime A.</creatorcontrib><creatorcontrib>Koo, Hyun</creatorcontrib><title>Extraction and purification of total RNA from Sreptococcus mutans biofilms</title><title>Analytical biochemistry</title><description>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
μg 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
μg 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.</description><subject>Biofilms</subject><subject>DNase I</subject><subject>Real-time PCR</subject><subject>RNA extraction</subject><subject>Streptococcus mutans</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><recordid>eNp1kEtLxDAURoMoOI7uXWblrvUm6SvuhmF8MSj4WIc0D8jQNjVJRf-9HcetcOGDy3cu3IPQJYGcAKmud7lscwpQ58ByoOQILQjwKgMG_BgtAIBltOL1KTqLcQdASFFWC_S4-UpBquT8gOWg8TgFZ52SvwtvcfJJdvjlaYVt8D1-DWZMXnmlpoj7Kckh4tZ567o-nqMTK7toLv5yid5vN2_r-2z7fPewXm0zxWiZMlaQGnTNOWkK1Ta2bUxDedsqo4wtKlMSy7RVRVXoWtdGc1mpeUpKCkqkJmyJrg53x-A_JhOT6F1UpuvkYPwUBeFNA5SVcxEORRV8jMFYMQbXy_AtCIi9NLETshV7aQKYmKXNyM0BMfMDn84EEZUzgzLaBaOS0N79D_8Andl0sg</recordid><startdate>20070615</startdate><enddate>20070615</enddate><creator>Cury, Jaime A.</creator><creator>Koo, Hyun</creator><general>Elsevier Inc</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20070615</creationdate><title>Extraction and purification of total RNA from Sreptococcus mutans biofilms</title><author>Cury, Jaime A. ; Koo, Hyun</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c325t-34170d799184cb8fb8e829bbcecef46e51f3dfc464d7d7ed9a6ca6c521421ad13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Biofilms</topic><topic>DNase I</topic><topic>Real-time PCR</topic><topic>RNA extraction</topic><topic>Streptococcus mutans</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Cury, Jaime A.</creatorcontrib><creatorcontrib>Koo, Hyun</creatorcontrib><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Cury, Jaime A.</au><au>Koo, Hyun</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Extraction and purification of total RNA from Sreptococcus mutans biofilms</atitle><jtitle>Analytical biochemistry</jtitle><date>2007-06-15</date><risdate>2007</risdate><volume>365</volume><issue>2</issue><spage>208</spage><epage>214</epage><pages>208-214</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><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
μg 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
μg 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.</abstract><pub>Elsevier Inc</pub><doi>10.1016/j.ab.2007.03.021</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
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| ispartof | Analytical biochemistry, 2007-06, Vol.365 (2), p.208-214 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_19880235 |
| source | ScienceDirect Journals |
| subjects | Biofilms DNase I Real-time PCR RNA extraction Streptococcus mutans |
| title | Extraction and purification of total RNA from Sreptococcus mutans biofilms |
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