Miscript nucleics mix

Reverse transcription was carried out using a miScript II RT Kit (Qiagen). A 20 μL reverse transcription reaction was prepared with 2.2 μL of eluted miRNA, 4 μL 5× miScript HiSpec Buffer (Qiagen), 2 μL 10× miScript Nucleics Mix (Qiagen), 9.8 μL RNase-free water, and 2 μL miScript Reverse Transcriptase Mix (Qiagen). The reaction was incubated at 16 °C for 60 min followed by 95 °C for 5 min. The reverse transcription reaction was then diluted with 200 μL RNase-free water. Triplicates of qPCR reactions were carried out using miScript SYBR Green PCR Kit (Qiagen) and run on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The reaction contained 2 μL diluted cDNA, 12.5 μL 2× QuantiTect^® SYBR Green PCR Master Mix (Qiagen), 2.5 μL 10× miScript Universal Primer (Qiagen), 10× miScript Primer Assay (Qiagen) for the target miRNA, and 5.5 μL RNase-free water in a final volume of 25 μL. The reaction mixtures were incubated for 15 min at 95 °C, followed by 45 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Standard curves were generated from a series of dilutions for the target miRNA (synthetic miScript miRNA Mimics from Qiagen) for each plate (Supplementary Fig. 13). Quantitation cycle (Cq) values were acquired and analyzed using StepOne™ Software v2.3 in accordance with the MIQE guidelines^{43 (link)}.