A novel real‐time polymerase chain reaction method for high throughput quantification of small regulatory RNAs

Abstract

SummaryMicroRNAs (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.

High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers

“…Besides the relative quantification of miRNA expression, the synthetic miRNA standards could also allow the absolute quantification of miRNA expression from total RNA samples. In contrast to the previously reported miRNA real-time PCR assays that were performed under standard thermocycling profiles of 45-75 sec per cycle (Chen et al 2005;Raymond et al 2005;Shi and Chiang 2005;SharbatiTehrani et al 2008;Yang et al 2009), our assay was capable of fast thermocycling (10 sec per cycle) without any modification of the reaction mixtures. The fast-cycling capability of this assay may, in part, be attributed to the short amplicon (<50 base pairs) generated by heminested primers and the rapid fluorescence acquisition of SYBR Green I without the necessity of probe hydrolysis.…”

“…1). Initially, we noticed that in a number of previous reports (Chen et al 2005;Raymond et al 2005;Shi and Chiang 2005;Duncan et al 2006;Varkonyi-Gasic et al 2007;Sharbati-Tehrani et al 2008;Yang et al 2009), the reverse PCR primers were designed to anneal directly to the sequences in the RT oligonucleotide. To achieve specificity, a unique miRNA-specific fluorescent probe is required to discriminate the targets from the nonspecific amplicons (Chen et al 2005).…”

“…Attempts have been made to improve miRNA detection without reliance on fluorescent probes (Raymond et al 2005;Shi and Chiang 2005;Sharbati-Tehrani et al 2008); however, these assays usually involved multiple sample processing steps (Shi and Chiang 2005) and suffered from a limited dynamic range of detection (Raymond et al 2005) and/or poor specificity against homologous miRNAs (Raymond et al 2005;Shi and Chiang 2005;Sharbati-Tehrani et al 2008). It has been noticed that a consensus strategy of these assays and some other TaqMan assays (Varkonyi-Gasic et al 2007;Yang et al 2009) is to use a universal or common reverse PCR primer and/or a fluorescent probe for amplification and detection of multiple miRNAs. In these assays, specificity of real-time PCR is only achieved by the forward PCR primer, which is not sufficient for discrimination of many homologous miRNAs.…”

Diagnostic and prognostic value of plasma level of microRNA-92a in acute myeloid leukemia

“…Several methods, such as northern blot [36], beadbased flow cytometry, [37] microarray, quantitative realtime PCR (qRT-PCR) [38], and deep sequencing [39] have been developed to measure miRNA expression. Of these methods, Mestdagh P et al [40] reported that qRT-PCR is superior due to its high sensitivity, specificity and reproducibility.…”

Evaluation of Three RT-qPCR-Based miRNA Detection Methods Using Seven Rice miRNAs

“…Because it is more sensitive, easier, and less time-consuming than approaches such as cloning, northern blotting, microarray analysis, and deep sequencing, a combination of reverse transcription (RT) and quantitative PCR, or RT-qPCR, is currently the most convenient and practical means of detecting precursor and mature miRNAs. Four well-developed RT-qPCR methods of miRNA detection, each with its own advantages and disadvantages, have recently become established: stem-loop RT-qPCR, 2) poly(A)-tailing RT-PCR, 3) primer-extension RT-qPCR 4) with various improvements, [5][6][7][8][9] and miQPCR. 10) Although stem-loop RT-qPCR is the most frequently used of these methods, in previous studies we were unable to detect certain rice miRNAs using it.…”