Open AccessA novel real‐time polymerase chain reaction method for high throughput quantification of small regulatory RNAs
Author(s) -
Yang Heping,
Schmuke Jon J.,
Flagg Lisa M.,
Roberts James K.,
Allen Ed M.,
Ivashuta Sergey,
Gilbertson Larry A.,
Armstrong Toni A.,
Christian Allen T.
Publication year - 2009
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/j.1467-7652.2009.00429.x
Subject(s) - biology , microrna , gene silencing , computational biology , small rna , small interfering rna , taqman , rna , gene expression , gene , gene expression profiling , microbiology and biotechnology , genetics , real time polymerase chain reaction
Summary MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are important players of both transcriptional and post‐transcriptional gene silencing networks. In order to investigate the functions of these small regulatory RNAs, a system with high sensitivity and specificity is desperately needed to quantitatively detect their expression levels in cells and tissues. However, their short length of 19–24 nucleotides and strong similarity between related species render most conventional expression analysis methods ineffective. Here we describe a novel primer for small RNA‐specific reverse transcription and a new TaqMan technology‐based real‐time method for quantification of small RNAs. This method is capable of quantifying miRNA and siRNA in the femtomolar range, which is equivalent to ten copies per cell or fewer. The assay has a high dynamic range and provides linear readout of miRNA concentrations that span seven orders of magnitude and allows us to discriminate small RNAs that differ by as little as one nucleotide. Using the new method, we investigated the expression pattern of gma‐miRMON1, a novel miRNA identified from soybean leaves. The results were consistent with our results obtained from Northern blot analysis of gma‐miRMON1 and Affymetrix microarray analysis of the gma‐miRMON1 precursor, suggesting that the new method can be used in transcription profiling.