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.
To make an internal control RNA, one needs to engineer a different beacon site into the amplicon. This can be accomplished by a combination of PCR and cloning.
The same primers are used to amplify the native (wild type) and IC-RNA, but unique beacons detect the each separately. One beacon is labeled with FAM, and the other with ROX, and amplification occurs simultaneously in real time. This enables correction for the slight variation in efficiency of amplification (tube-to-tube variation) or that incurred by inhibitors.
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Resulting standard curves have higher coefficients of determination (r2) values.
IC-NASBA standard curve for K. brevis.
Normal time-to-positivity determination of standard curve values.
Reference: Patterson, S.S., E. T. Casper, L. Garcia-Rubio, M. C. Smith and J. H. Paul. 2005. Increased Precision of Microbial RNA Quantification using NASBA with an Internal Control (IC-NASBA). J. Microb. Methods (in press).