Tiangen total rna extraction kit

To validate the transcriptome data, the relative expression of nine DEGs identified in the transcriptome analysis was assessed by performing qRT-PCR, using three biological and three technical replicates. We performed RNA extraction and cDNA synthesis using the TIANGEN Total RNA Extraction Kit (TIANGEN, Beijing, China) and PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), respectively. The specific primers for selected DEGs were designed using the Primer premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and are shown in (Supplementary Table S1). The actin gene was used as an internal control. qRT-PCR detection was performed on the ABI 7500 Fast Real-Time PCR System using the TaKaRa SYBR Green Master Mix Kit (TaKaRa, Beijing, China).

To validate the transcriptome data, the relative expressions of 12 DEGs identified in transcriptome analysis were evaluated by qRT-PCR using three biological and three technical replicates in the analysis. RNA extraction and cDNA synthesis were conducted using the Tiangen total RNA extraction kit (Tiangen, Beijing, China) and PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Kyoto, Japan), respectively. The primers for qRT-PCR were designed using the Primer Premier 5 software (http://www.premierbiosoft.com/primerdesign/index.html) (Accessed 20 April 2021). qRT-PCR tests were carried out with the TaKaRa SYBR Green Mix kit (TaKaRa, Beijing, China) using the ABI 7500 Fast Real-Time Detection System. PCR amplification was performed in 20 µL containing 10 µL 2xSYBR Primix ExTaq, 0.8 µL upstream primer, 0.8 µL downstream primer, 0.4 µL Rox-Reference dye, 2 µL cDNA template and 6 µL ddH₂O under a standard PCR program of 95 °C for 30 s; 40 cycles at 95 °C for 5 s; 60 °C for 35 s; 95 °C for 15 s; and 60 °C for 1 min, followed by 95 °C for 15 s. The relative expression analysis of quantitative data was performed by using 2^−ΔΔCt with 18S-rRNA as the reference gene.