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

Abstract

MicroRNAs are small noncoding RNAs that serve as important regulators of eukaryotic gene expression and are emerging as novel diagnostic and therapeutic targets for human diseases. Robust and reliable detection of miRNAs is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Existing methods for miRNA quantification rely on fluorescent probes for optimal specificity. In this study, we developed a highperformance real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay that allows specific and rapid detection of mature miRNAs using a fast thermocycling profile (10 sec per cycle). This assay exhibited a wide dynamic range (>7 logs) and was capable of detecting miRNAs from as little as 1 pg of the total RNA or as few as 10 cells. The use of modified reversetranscription oligonucleotides with a secondary structure and hemi-nested reverse PCR primers allowed excellent discrimination of mature miRNAs from their precursors and highly homologous family members using SYBR Green I. Using a novel approach involving uracil-DNA glycosylase treatment, we showed that carryover of the reverse transcription oligonucleotide to the PCR can be successfully eliminated and discrimination between miRNA homologs could be further enhanced. These assays were further extended for multiplexed detection of miRNAs directly from cell lysates without laborious total RNA isolation. With the robust performance of these assays, we identified several miRNAs that were regulated by glial cell-line-derived neurotrophic factor in human glioblastoma cells. In summary, this method could provide a useful tool for rapid, robust, and cost-effective quantification of existing and novel miRNAs.

Pilot Study of Serum MicroRNA-21 as a Diagnostic and Prognostic Biomarker in Egyptian Breast Cancer Patients

“…This method was described by Schmittgen et al as the most sensitive, specific, fast, and efficient method, which only needs small amounts of RNA for analysis, and could even differentiate between miRNAs that differ by only one or two base pairs [23]. We measured the active mature miR-21 in serum, rather than the inactive precursor, since it reflects the regulation of both miRNA processing and maturation, especially in various human disease where alterations in miRNA biogenesis produce levels of mature miRNA that are different from those of the pre-miRNA [22,27]. RNU6B, used as an endogenous control, was consistently expressed in all study groups and was not influenced by BC status, and this was in accordance with multiple studies which used RNU6B as a normalizer in cancer [1,7,28,29].…”

Molecular Basis for Interaction of let-7 MicroRNAs with Lin28

“…Briefly, pri-let-7g was co-transfected with FLAG-tagged Lin28 constructs (25ng unless otherwise noted) or vector control into 293T cells (12well) using lipofectamine. Total RNA was isolated using TriZol reagent, treated with DNAse I, and quantitative RT-PCR was used with miRNA-specific stem-loop primers as previously described (Wan et al, 2010). Relative levels of mature miRNAs were analyzed by ΔΔCt method, and normalized by U6 snRNA levels.…”

miR-ID: A novel, circularization-based platform for detection of microRNAs

“…In this respect, TaqMan microRNA assays rely on the higher affinity of their stemloop RT primer for the 39-end of the mature miRNA sequence than for the same sequence within the hairpin structure of the corresponding pre-miRNA form . The extent of such affinity-based discrimination varies dramatically depending on pre-miRNA structure and sequence Wan et al 2010). In contrast, miR-ID can reliably discriminate these two miRNA forms at both RT and PCR steps by using primers that cross boundaries between miRNA sequences repeats targeting specifically one form over another.…”

“…Among the latter methods, the TaqMan microRNA assay, which uses stem-loop RT primers together with miRNA-specific TaqMan probes, is often considered as the ''gold standard'' for microRNA detection ABI 2010). Modified versions of the TaqMan assay that use an alternative primer design and target-specific probes have also been described recently (Lao et al 2006;Yang et al 2009;Varkonyi-Gasic and Hellens 2010;Wan et al 2010). Other available RT-qPCR miRNA assays use a single nonspecific dye such as SYBR Green rather than miRNA-specific TaqMan probes (Raymond et al 2005;Shi and Chiang 2005;Ro et al 2006;SharbatiTehrani et al 2008;Wang 2009;Varkonyi-Gasic and Hellens 2010).…”