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RNA diagnostics: real‐time RT‐PCR strategies and promising novel target RNAs

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Abstract Ribonucleic acid (RNA) is a multifunctional type of molecule, playing critical roles in protein biosynthesis and regulation. In recent years, suppression of protein translation by so‐called microRNAs came into the focus of research, especially because deregulation of this process has been shown to play a role in malignant transformation. Furthermore, RNA molecules circulating in the blood have been revealed as a novel class of markers for diagnosis of cancers. Moreover, genetic information of some pathogens is stored as RNA, allowing their sensitive detection using nucleic acid amplification techniques. In this article, the principle of detecting different RNA types by real‐time reverse‐transcription polymerase chain reaction applications is described. Furthermore, the emerging use of microRNA and circulating RNA profiles complementing the broad spectrum of RNA diagnosis is discussed. WIREs RNA 2011 2 32–41 DOI: 10.1002/wrna.46 This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses In Vitro and In Silico

MicroRNA biogenesis. MicroRNAs are generated in viv from primary transcripts, produced by RNA polymerase II52,53 or III,54 either from separate transcription units or from clustered units forming polycistronic transcripts (1). In the nucleus these long ribonucleic acids, referred to as primary microRNAs (pri‐miRNAs), are cleaved by the microprocessor complex into 60–70 nt stem‐loop intermediates (2), known as precursor microRNAs, or pre‐miRNAs.20,55 The microprocessor complex56 consists of Drosha57 and its cofactor, the DGCR8 protein.25 The pre‐miRNA is shuttled out of the nucleus by Exportin‐5, a nuclear export factor58,59 (3). Once in the cytoplasm the pre‐miRNA is cut into an imperfect 21 nt duplex by Dicer,57 and its cofactors (4). The resulting imperfect duplex, consisting of the miRNA strand (guide strand) and the miRNA* strand (passenger strand), is unwound and integrated into the RNA‐induced silencing complex (RISC)60 (5 & 6). The decision which one of the strands acts as guide strand was found to be dependent on the internal stability of the miRNA duplexes.61 The miRNA serves as an adapter to guide the RISC enzyme complex to the 3′ untranslated region (3′ UTR) of the target mRNA (6), either enabling translational inhibition or leading to mRNA degradation62 (7 & 8).

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Detection of mature miRNA by MGB probe‐based real‐time‐RT‐PCR. (a) Stem‐loop RT primers bind to the 3′ end of the mature miRNA molecules, initiating reverse transcription. (b) Subsequently, cDNA is amplified using target specific (5′‐elongated) forward primers (FW) and universal reverse primers (Rev) targeting the introduced stem‐loop‐sequence. Target‐specific fluorescence generation is mediated by the cleavage of short 5′ nuclease probes bearing a MGB‐molecule () at their 3′‐end. For details on the principle of 5′ nuclease probe‐mediated fluorescence generation see Figure 1.

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Real‐time‐PCR kinetics and multiplexed applications. (a) ½ log10‐dilution series of porphobilinogen desaminase (PBGD)‐mRNA used as calibrators for the quantification of unknowns. The 40 observations of relative change in fluorescence intensity show the typical sigmoid trajectories of the amplification reactions from an early exponential phase to a late plateau phase. The point at which amplification‐derived fluorescence generation becomes significantly detectable and passes a certain threshold (shown as the horizontal line) is called the Ct‐ or CP‐value and is used for the generation of a corresponding standard curve (inlet). (b) Parallel detection of plasma HIV‐1‐RNA and an artificial internal control RNA in a multiplex one‐step real‐time RT‐PCR configuration. 5′ nuclease probes labeled with FAM or VIC fluorescent reporter dyes were used for specific detection of HIV‐1‐genomes or internal controls, respectively. Only samples negative for FAM‐ but positive for VIC‐mediated fluorescence generation are reliably tested ‘nonreactive’ for HIV‐1‐RNA.

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Fluorogenic probe chemistries. (a) 5′ nuclease probes. Downstream of one of the primers, additionally a dual‐labeled hybridization probe anneals to the DNA target molecule. During primer extension the 5′ → 3′ exonuclease activity of Taq DNA polymerase hydrolyzes the probe, separating a fluorescent reporter dye () from an intramolecular quencher (), giving rise to specific fluorescence emission. (b) Scorpion primers. After annealing, the primer section is extended by the DNA polymerase. After strand separation, the probe section of the Scorpion oligodeoxynucleotide hybridizes to a region downstream from the primer sequence during the annealing step of the PCR reaction. The hairpin structure of the Scorpion primer is blocked from extension () to ensure that the reporter dye () and quencher () are only separated by specific hybridization of the probe section to the target sequence. (c) Adjacent hybridization probes. Two linear fluorogenic oligoprobes hybridize next to each other to the target sequence during the annealing step of the PCR reaction. The acceptor dye () then emits fluorescence due to corresponding excitation by the excited donor dye () in close proximity. (d) Molecular beacons. Hybridization of the probe sequence to the target sequence during the annealing step separates the reporter dye () from the quencher ().

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RNA Methods > RNA Analyses In Vitro and In Silico
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
RNA in Disease and Development > RNA in Disease

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