Real-time quantification of microRNAs by stem–loop RT–PCR

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 efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related mi...

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Bibliographic Details
Published in:	Nucleic acids research Vol. 33; no. 20; p. e179
Main Authors:	Chen, Caifu, Ridzon, Dana A., Broomer, Adam J., Zhou, Zhaohui, Lee, Danny H., Nguyen, Julie T., Barbisin, Maura, Xu, Nan Lan, Mahuvakar, Vikram R., Andersen, Mark R., Lao, Kai Qin, Livak, Kenneth J., Guegler, Karl J.
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 27-11-2005 Oxford Publishing Limited (England)
Subjects:	Animals Cell Line DNA Primers - chemistry Humans Methods Online Mice MicroRNAs - analysis MicroRNAs - metabolism Nucleic Acid Conformation Reverse Transcriptase Polymerase Chain Reaction - methods RNA Precursors - analysis
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Summary:	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 efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. 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 variation 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.
Bibliography:	To whom correspondence should be addressed. Tel: +1 650 638 5245; Fax: +1 650 638 6343; Email: chencx@appliedbiosystems.com local:gni178 ark:/67375/HXZ-BRJWDQGX-Q istex:3356507E3E7FF1C0DAC8F514766A9C7EAE1E42F3 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/gni178