title: Increased precision of microbial RNA quantification using NASBA with an internal control.
abstract: Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.
authors.affiliation: College of Marine Science, University of South Florida, 140 7th Ave., South, St. Petersburg, FL 33701, USA.
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Patterson
authors.forename: Erica T
authors.initials: ET
authors.lastname: Casper
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia-Rubio
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Smith
authors.forename: John H
authors.initials: JH
authors.lastname: Paul
mesh_terms: D000818:Animals; D001483:Base Sequence; D004141:Dinoflagellida / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards*; D016053:RNA, Protozoan / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D016053:RNA, Protozoan