Real time pcr reagents

The reverse transcription reaction was performed using the miScript reverse transcription kit (Qiagen), according to the manufacturer’s instructions. Real-time PCR reagents and miScript primers for miRNAs were from Qiagen. Amplification reactions were performed using a StepOne Plus real-time PCR system (Applied Biosystems), according to the manufacturer’s manual; each reaction had three technical replicates, and data are presented as means ± standard deviation.
For normalization purposes, we used an adaptation of the normalization procedure used in [25 (link)]. For each sample, we computed which percentage of the total amount of RNA extracted in the IP experiments corresponded to the amount of RNA used in the RT-qPCR. Input RNA was used as the reference RNA. For each IP sample, a normalization factor was computed by dividing the percentage of RNA in the IP sample by the percentage of RNA in the Input sample. After RT-qPCR, for each miRNA, IP results were first compared with the Input RNA, then divided by the respective normalization factor. Differences between IP samples and IgG controls were calculated based on the 2^−ΔΔC(t) method.