title: A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA.
abstract: Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem-loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem-loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Jung
authors.forename: Xiaoou
authors.initials: X
authors.lastname: Jiang
authors.forename: Stefan H E
authors.initials: SH
authors.lastname: Kaufmann
authors.forename: Volker
authors.initials: V
authors.lastname: Patzel
mesh_terms: D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000191:economics / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D008089:Listeria monocytogenes / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; C548234:MIRN133 microRNA, human; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated
keywords: TaqMan PCR; noncoding RNA detection; real-time PCR; stem–loop primer