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Detection and analysis of spliced chimeric mRNAs in sequence databanks

Nucleic Acids Research
Oxford University Press
Publication Date
  • Nar Methods Online
  • Biology
  • Design
  • Medicine


gni178 1..9 Real-time quantification of microRNAs by stem–loop RT–PCR Caifu Chen*, Dana A. Ridzon, Adam J. Broomer, Zhaohui Zhou, Danny H. Lee, Julie T. Nguyen, Maura Barbisin, Nan Lan Xu, Vikram R. Mahuvakar, Mark R. Andersen, Kai Qin Lao, Kenneth J. Livak and Karl J. Guegler Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA Received May 24, 2005; Revised July 8, 2005; Accepted October 25, 2005 ABSTRACT A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT effici- ency and specificity. TaqMan miRNA assays are spe- cific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleot- ide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed vari- ation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency. INTRODUCTION MicroRNAs (miRNAs) are naturally occurring, highly con- served families of transcripts (18–25 nt in length) that are processed from larger hairpin precursors (1,2). miRNAs are found in the genomes of animals (3–9) and

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