Analyses of miRNA/messenger RNA (mRNA) expression were assessed as previously described.14 (link),21 (link) Briefly, for miRNA studies, single miRNA assays for real-time reverse transcription–PCR (RT-PCR) (Applied Biosystems, Zug, Switzerland) were used. Megaplex Primer Pool A v2.1 (Applied Biosystems) was used for RT-PCR according to manufacturer's recommendations. Expressions of POU2AF1 and Spi-B were analyzed with quantitative real-time RT-PCR by using Assay-On-Demand reagents (Applied Biosystems). As previously validated,20 (link) RNU44 miRNA and PUM1 mRNA were used as references for normalization and relative expression analyses. The comparative cycle threshold method (Applied Biosystems) was used for calculations of relative quantitation of targets. Quantitative PCR probe sequences for miRNAs and mRNAs are presented in table e-1 at Neurology.org/nn .
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Quantitative Analysis of miRNA and mRNA
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Other organizations : Ruhr University Bochum, St. Josef-Hospital
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Variable analysis
independent variables
- MiRNA assays for real-time reverse transcription–PCR (RT-PCR)
- Megaplex Primer Pool A v2.1 for RT-PCR
- Quantitative real-time RT-PCR for POU2AF1 and Spi-B expressions
- MiRNA expressions
- Expressions of POU2AF1 and Spi-B
- RNU44 miRNA and PUM1 mRNA as references for normalization and relative expression analyses
- Previously validated RNU44 miRNA and PUM1 mRNA as positive controls
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