Sensitive multiplex RNA quantification using capillary electrophoresis‐based single‐strand conformation polymorphism

Abstract

Quantification of RNA provides information crucial for various biological studies, including analysis of mRNA expression and that of microRNAs. Reverse transcription (RT) coupled with real-time polymerase chain reaction (PCR) is known to be the most accurate method for quantifying nucleic acids, and thus represents the state-of-the-art for RNA quantification. However, the use of real-time PCR for RNA quantification is limited to a single target per analytical run because of reductions in quantification power and limitations of fluorescence dyes associated with multiplex applications. Here, we report a novel multiplex RNA quantification method that uses capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) coupled with modified RT and asymmetric PCR. The reverse transcripts of seven in vitro transcribed RNAs were modified with common sequence tags and amplified by asymmetric PCR using primers specific to the common tags. The resulting amplicons were separated and quantified by CE-SSCP. A series of experiments using different amounts of RNA demonstrated that the assay had a limit of detection of 2 amol and a dynamic range of approximately 10(5). These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification.

Determination of Baylisascaris schroederi Infection in Wild Giant Pandas by an Accurate and Sensitive PCR/CE-SSCP Method

“…PCR products from the adult B. schroederi and one or two positive PCRs per fecal sample were analyzed with the CE-SSCP as described by Park et al [16] and Shin et al [17] . Park et al [16] found that high concentration DNA templates might result in nonlinear correlation between the peak areas of CE-SSCP and DNA amounts.…”