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Title: A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA
Authors: Jung, Ulrike
Kaufmann, Stefan H E
Volker Patzel 
Keywords: TaqMan PCR
noncoding RNA detection
real-time PCR
stem–loop primer
DNA Primers
DNA Probes
Escherichia coli
Fluorescent Dyes
Gene Expression
Gene Expression Profiling
HEK293 Cells
Inverted Repeat Sequences
Listeria monocytogenes
RNA, Bacterial
RNA, Small Untranslated
Real-Time Polymerase Chain Reaction
Reverse Transcriptase Polymerase Chain Reaction
Sensitivity and Specificity
Issue Date: 1-Dec-2013
Publisher: Cold Spring Harbor Laboratory Press
Source: Jung, Ulrike, JIANG XIAOOU, Kaufmann, Stefan H E, Volker Patzel (2013-12-01). A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA. RNA 19 (12) : 1864-1873. ScholarBank@NUS Repository.
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.
Source Title: RNA
ISSN: 13558382
DOI: 10.1261/rna.040501.113
Appears in Collections:Staff Publications

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