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Sensitive multiplex RNA quantification using capillary electrophoresis‐based single‐strand conformation polymorphism
Author(s) -
Shin Gi Won,
Hwang Hee Sung,
Nam Hong Gil,
Oh MiHwa,
Jung Gyoo Yeol
Publication year - 2010
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22646
Subject(s) - multiplex , rna , amplicon , capillary electrophoresis , microbiology and biotechnology , biology , nucleic acid , multiplex polymerase chain reaction , dna , rna extraction , real time polymerase chain reaction , computational biology , polymerase chain reaction , chemistry , genetics , gene
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 ∼10 5 . These results clearly indicate the potential of this method to provide robust and precise multiplex RNA quantification. Biotechnol. Bioeng. 2010; 106: 167–172. © 2009 Wiley Periodicals, Inc.