Ssofast evagreen sybr green qpcr master mix

Total RNA was extracted from cells or tissues using Trizol (ThermoFisher) according to the manufacturer’s manuals. RNA samples were quantified and qualified using NanoDrop analysis (ThermoFisher) and 1% agorose gels. Those with sufficient concentration (>30 ng/µl), purity (A260/280 > 1.7) and integrity (clear 28 and 18s rRNA bands) were converted to cDNA using a high capacity cDNA reverse transcription kit (ThermoFisher) following the manufacturer’s protocol. The levels of miR-125a and miR-125b were quantified using Taqman^® miRNA assays (ThermoFisher), normalized to the expression of U6b snRNA, or using the miRCURY™ LNA™ Universal RT microRNA PCR (Qiagen, Germany), normalized to the expression of miR-103a. Anti-miR-125b ASOs was quantified using a Taqman^® miRNA assay (ID #007655_mat), normalized to the expression of U6b snRNA or its copy number was calculated based on a standard curve of ASOs threshold cycle (Ct) values vs. the concentration of the ASOs in a serial dilution from 0.001 to 1000 pM. qRT-PCR analysis of mRNAs was performed using Ssofast^TM Evagreen (SYBR Green) qPCR master mix (Bio-Rad), normalized to the expression of GAPDH, or 18s rRNA, or ACTB. All qPCR reactions were performed by using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad).

Total RNA was extracted, cleaned and concentrated from cells using RNase Plus Mini Kit (Qiagen, Germany) according to the manufacturer's manuals. RNA samples were quantified using NanoDrop analysis (Thermo Fisher Scientific, USA). 100 ng total RNA was converted to cDNA with the Superscript III first-strand synthesis system (200 U μL^–1) (Thermo Fisher Scientific, USA) by reverse transcription. qRT-PCR analysis of mRNAs was performed using an SsofastTM Evagreen (SYBR Green) qPCR master mix (Bio-Rad). All qPCR reactions were performed by using a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA).