{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T01:35:08Z","timestamp":1790386508456,"version":"4.1.0"},"reference-count":43,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2005,7,1]],"date-time":"2005-07-01T00:00:00Z","timestamp":1120176000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2005,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (\n            <jats:italic>ref<\/jats:italic>\n            mRNA) along with mRNA absolute standard curves. The technique was applied to quantify mRNA of the trichloroethene (TCE) reductive dehalogenase-encoding\n            <jats:italic>tceA<\/jats:italic>\n            gene in an anaerobic TCE-to-ethene dechlorinating microbial enrichment. Compared to RT-qPCR protocols that utilize DNA absolute standard curves, application of the new technique increased measured quantities of\n            <jats:italic>tceA<\/jats:italic>\n            mRNA by threefold, demonstrating a substantial improvement in quantification. The technique was also effective for quantifying the loss of mRNA during specific steps of the sample processing protocol. Analysis revealed that the efficiency of the RNA isolation (56%) step was significantly less than that of the cell lysis (84%), DNA removal (93%), and RT (88%) steps. The technique was applied to compare the effects of cellular exposure to different chlorinated ethenes on\n            <jats:italic>tceA<\/jats:italic>\n            expression. Results show that exposure to TCE or\n            <jats:italic>cis<\/jats:italic>\n            -1,2-dichloroethene resulted in 25-fold-higher quantities of\n            <jats:italic>tceA<\/jats:italic>\n            mRNA than exposure to vinyl chloride or chlorinated ethene starvation.\n          <\/jats:p>","DOI":"10.1128\/aem.71.7.3866-3871.2005","type":"journal-article","created":{"date-parts":[[2005,7,7]],"date-time":"2005-07-07T06:37:11Z","timestamp":1120718231000},"page":"3866-3871","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":145,"title":["An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the\n            <i>tceA<\/i>\n            Reductive Dehalogenase Gene"],"prefix":"10.1128","volume":"71","author":[{"given":"David R.","family":"Johnson","sequence":"first","affiliation":[{"name":"Department of Civil and Environmental Engineering, University of California, Berkeley, California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick K. 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