Computational protocol: Assessment of RT-qPCR Normalization Strategies for Accurate Quantification of Extracellular microRNAs in Murine Serum

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Protocol publication

[…] All RT-qPCR studies were designed to comply with the MIQE guidelines where applicable or practical , (). Small RNA TaqMan Assays (Life Technologies) were performed as according to manufacturer’s instructions as described previously . Serum RNA samples were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) using miRNA-specific stem-loop RT. qPCR analysis was performed on a StepOne Plus real-time thermocycler using TaqMan Gene Expression Mastermix (Life Technologies). All primer/probe assays are listed in . . TaqMan assays were validated by performing serial dilutions of cDNA to produce standard curves in order to demonstrate assay linearity and dynamic range (). PCR efficiencies were determined by linear regression analysis performed directly on the sample data using LinRegPCR (). Data were analyzed using the Pfaffl method in order to correct for PCR efficiency . [...] miRNA expression stability was determined using the Normfinder Visual Basic application for Microsoft Excel . Correlation analyses, one-way analysis of variance (ANOVA) or two-way ANOVA (with Bonferroni post hoc tests) as appropriate (GraphPad Software Inc, La Jolla, CA, USA). […]

Pipeline specifications

Software tools LinRegPCR, NormFinder
Application qPCR
Organisms Mus musculus
Diseases Muscular Dystrophies, Muscular Dystrophy, Duchenne