Validating miRNA Expression by RT-qPCR

To validate the differential miRNA expression, RT-qPCR was performed as described previously [17 (link)]. First, 10 ng of total RNA was reverse-transcribed using a multiplex approach with TaqMan primers (reference gene: RNU48 (Assay ID: 001006) or RNU44 (Assay ID: 001094), ssc-miR-335-5p (Assay ID: 244560_mat) and hsa-miR-335* (hsa-miR-335-3p, Assay ID:002185); Applied Biosystems, Carlsbad, CA, USA) [70 (link)]. Subsequently, qPCR was done using TaqMan microRNA assays (Applied Biosystems) and a LightCycler®480 Probes Master (Roche Diagnostics GmbH, Mannheim, Germany).
The reactions were run in triplicate on the LightCycler®480 Real-Time PCR system (Roche Diagnostics GmbH). The LightCycler®480 software release 1.5.0 (Roche Diagnostics GmbH) was used to analyze the data. The relative miRNA expression levels were calculated using the formula 2^−ΔCp.

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Ong J., van den Berg A., Faiz A., Boudewijn I.M., Timens W., Vermeulen C.J., Oliver B.G., Kok K., Terpstra M.M., van den Berge M., Brandsma C.A, & Kluiver J. (2019). Current Smoking is Associated with Decreased Expression of miR-335-5p in Parenchymal Lung Fibroblasts. International Journal of Molecular Sciences, 20(20), 5176.

Publication 2019

TaqMan primers TaqMan microRNA assays LightCycler®480 Probes Master LightCycler®480 Real-Time PCR system LightCycler®480 software release 1.5.0

Assays Diagnostics Gene Hsa mir 335 Mirna Primers

Corresponding Organization : University Medical Center Groningen

Other organizations : University of Technology Sydney, Woolcock Institute of Medical Research, University of Sydney

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Variable analysis

independent variables

Reverse transcription using a multiplex approach with TaqMan primers

dependent variables

Relative miRNA expression levels calculated using the formula 2^−ΔCp

control variables

Reference gene: RNU48 (Assay ID: 001006) or RNU44 (Assay ID: 001094)
Amount of total RNA used for reverse transcription: 10 ng

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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