Tb green premix ex taq 2 til rnaseh plus

Briefly, total RNA was extracted from each cell sample receiving the NaCr treatment (treatment condition in Section 2.8) using RNAiso Plus Reagent (Takara, Kyoto, Japan) and reverse-transcribed using the PrimeScript RT-PCR kit with gDNA Eraser (Takara, Kyoto, Japan). The cDNA templates were tested for the absence of contaminated genomic DNA using GAPDH primers that amplify different fragment sizes with genomic DNA and cDNA templates (Supplementary Figure S2). The obtained cDNA was used as a template for qRT–PCR. qRT-PCR reactions were performed with TB Green Premix Ex Taq II (Til RNaseH Plus, Takara, Kyoto, Japan) and run on a CFX96 (Bio-Rad) system. The primers used are listed in Table 1. The relative mRNA levels were analyzed using the 2^−ΔΔCt method (normalized with GAPDH).

Total RNA was extracted with RNAiso Plus (#9109, TakaRa Bio Inc, Shiga, Japan) as previously described. RNA was reverse transcribed into cDNA with PrimeScript RT reagent Kit (#RR037, TakaRa Bio Inc). cDNA was then amplified with TB green Premix Ex Taq II (Til Rnase H Plus) (##RR820A, TakaRa Bio Inc). The real-time PCR was conducted with a LightCycler 96 RT-qPCR System (Roche, Basel, Switzerland). The relative quantity of the targeted RNA was calculated through normalization to the quantity of the corresponding GAPDH mRNA level. Detailed primer sequences are listed in Supplementary file 2.