Primesript rt reagent kit

Total RNA from each of the E- and CON-piPSCs was extracted by using Trizol reagent (TaKaRa, Dalian, China) according to the manufacturer’s protocol. The cDNA was synthesized by reverse transcription PCR using the PrimeSript™ RT reagent Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The qRT-PCR reaction system included, in volume: 10 μL of SYBR^® Premix Ex Taq II (Tiangen) (2×), 1 μL of cDNA, and 1 μL of PCR Forward/Reverse Primer (10 μmol/L), and RNase-free water was added to achieve a total volume of 20 μL. The specific primers of genes used in this study are shown in Table 1. Relative mRNA expression levels of these genes for each treatment were normalized by the geometric mean of β-actin and were computed by the 2^−ΔΔCT method [25 (link)]. β-ACTIN was used as an endogenous loading control.

Total RNA was extracted by using an RNAiso Plus reagent (AL11817A, TaKaRa, Beijing, China) according to protocol. The total RNA extracted from CHIR- and sh-AXIN2CHIR- piPSCs were collected after the CHIR99021 withdrawal for three passages, and the total RNA extracted from sh-AXIN1, sh-AXIN2, OE-AXIN1, and OE-AXIN2 ipPSCs were collected after cell line purification. Then, by using the PrimeSript™ RT reagent Kit (Tiangen, China) according to its protocol, the cDNA was synthesized as a reverse transcription PCR (RT-PCR). The qRT-PCR reaction system was 20 μL in volume: 10 μL SYBR^® Premix Ex Taq II (Vazyme), 0.5 μL cDNA, 0.4 μL PCR Forward Primer (10 μM), 0.4 μL PCR Reverse Primer (10 μM), and RNase-free water added to a total volume of 20 μL.