Mircury lna uni rt primer mix

Total RNA was extracted from mouse and human tissue and mouse-derived cultured cell lines using TRIzol reagent from the Direct-Zol™ RNA Miniprep kit (R2051) following the manufacturer’s protocol. Total RNA was reversely transcribed using a miScript II RT Kit (218161 Qiagen, Hilden, Germany), while mRNA templates were reversely transcribed using the Moloney Murine Leukemia Virus (M-MLV RT) reverse transcriptase by Promega (9PIM170; Promega, Madison, WI, USA). RT-PCR was performed using SYBR green, carried out on a Bio-Rad CFX96 Real-Time System. For validation of transfection efficiency in cell culture experiments, miR-337-3p pre-designed miRCURY LNA Uni RT primer mix (339306, Qiagen) was used. Consequently, total RNA was reversely transcribed using its compatible miRCURY LNA™ Universal cDNA synthesis kit II (203301, Qiagen) followed by real-time PCR with the LNA enhanced primers using ExiLENT SYBR green (203401, Thermofisher, Waltham, MA, USA). Quantification of transcript level by RT-PCR was done by using the relative Ct (ΔΔCt) method. mRNA transcripts were normalized to L7, while miR transcripts were normalized to U6 or 5s (pre-designed miRCURY LNA Uni RT primer mix, Qiagen). The choice of reference genes was based on their stability in the different samples analyzed. The sequence of the primers used in this study can be found in Table 1.