Miscript mirna pcr array human cancer pathway kit

For RNA immunoprecipitation (RIP), two rounds using 5 μg of anti-m6A (#ab208577, Abcam, Paris, France), anti-m7G (#6655, Biovison, Milpitas, CA, USA), and anti-m5C (#61255, Active Motif, Nantes, France) antibodies and 5 μg of small RNA were performed. The reaction was carried out using a Dynabeads protein G IP kit with some modifications (#10007D, Thermo Fisher Scientific, Paris, France) such as described by Berulava et al. (2015) [14 (link)] and Briand et al. (2021) [19 (link)]. As a control, IP was performed using IgG (#ab18443, Abcam, Paris, France) instead of an anti-m6A antibody. miRNAs obtained from RIP were reverse transcribed using a miScript II RT kit (#218161, Qiagen, Paris, France) and analyzed using the miScript miRNA PCR array human cancer pathway kit (#331221, Qiagen, Paris, France) according to the manufacturer’s instructions. Fold enrichment was next calculated using the Ct value obtained from qRT-PCR performed with input miR, IP-IgG, IP-m6A, and the 2^−ΔΔCt formula.

For IP of RNA, two rounds using 5 μg of anti-m6A antibody (Abcam, France) and 5 μg of small RNA were performed. The reaction was carried out using a Dynabeads protein G IP kit with some modifications (Thermo Fisher Scientific, France) such as described by Berulava et al.⁶ (link) As a control, IP was performed using IgG (Abcam, France) instead of anti-m6A antibody. miRNAs obtained from m6A IP were reverse transcribed using miRScript II RT kit (QIAGEN, France) and analyzed using the miScript miRNA PCR array human cancer pathway kit (QIAGEN, France) according to the manufacturers’ instructions. Fold enrichment was next calculated using Ct value obtained from qRT-PCR performed with input miR, IP-IgG, and IP-m6A, and the 2^−ΔΔCt formula.