Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs

There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcr...

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Bibliographic Details
Published in:RNA (Cambridge) 2005-11, Vol.11 (11), p.1737-1744
Main Authors: Raymond, Christopher K, Roberts, Brian S, Garrett-Engele, Phillip, Lim, Lee P, Johnson, Jason M
Format: Article
Language:English
Subjects:
DNA Primers
DNA, Complementary - metabolism
Gene Expression Profiling
Humans
Method
MicroRNAs - analysis
MicroRNAs - genetics
MicroRNAs - metabolism
Polymerase Chain Reaction - methods
Reverse Transcription
RNA, Small Interfering - analysis
RNA, Small Interfering - genetics
Tissue Distribution
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Summary:There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.2148705