Tb green premix ex taq 2 tli rnaseh plus reagent

Total RNA of mouse forelimbs and mBMSCs was extracted using an Eastep Super total RNA extraction kit (Promega, Shanghai, China) according to the manufacturer’s instructions and then was reverse transcribed using a PrimeScriptTM RT Master Mix (Takara Bio, Ohtsu, Japan). The quantification was performed using the TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) reagent (Takara Bio, Ohtsu, Japan). The relative expression of target gene was normalized in relative to the level of GAPDH using the 2^−ΔΔCT method. Primer sequences were provided in the Table 1.

RNA samples were extracted from EGFP-AUF1 p45-overexpressing HeLa and control cells. Cells were lysed with TRIzol (Invitrogen, Cat No. 15596026, USA), followed by isopropanol precipitation and removal of salts with 75% v/v ethanol/H₂O. We further removed traces of genomic DNA from the samples by gEraser treatment and performed reverse transcription with the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, RR047A, Beijing, China). To validate gene-specific total mRNA levels, we used TB Green Premix Ex Taq™ II (Tli RNaseH Plus) reagent (Takara, Cat No. RR820 L, Beijing, China). We collected the melting curves on a StepOne Plus Real-Time PCR System (Applied Biosystems 7500, USA) and analyzed the data on the instrument's software version 2.2.2.