Reverse aid first strand cdna synthesis kit

Transcript levels of genes associated with DEPs were determined using real-time quantitative polymerase chain reaction (RT-qPCR). For total RNA extraction, extract stems using an RNA Rapid Extraction Kit (Aidlab Biotech, Beijing, China) according to the operation manual. Use the Reverse Aid First Strand cDNA Synthesis Kit (TaKaRa Biotech, Beijing, China) to reverse-transcribe RNA to cDNA. The process was as follows: Mix RNA (2 μg) with 1 μL Oligo d (T) 18 (0.5 μg/μL), 2 × TS Reaction Mix (10 μL) and TransScript RT/RI Enzyme Mix (1 μL) with an extra 20 μL of RNase-free Water. Mix the mixture gently and incubate at 42 °C for 15 mins. Terminate the reaction by incubation at 85 °C for 5 s, and store the cDNAs of the product at − 20 °C. Use the cDNA samples as a template, and then mix them with 200 nmol primer and SYBR Green PCR Real Master Mix (TakaRa, Kusatsu, Japan) for real-time PCR analysis using Bio-Rad CFX 96 real-time PCR instruments and CFX manager software ver 3.0 (Bio-Rad Laboratories, California, USA). The PCR temperature procedure is as follows: 95 °C of predegeneration for 3 mins, 40 cycles of denaturation at 95 °C for 20 s, annealing at 59 °C for 20 s, and extension at 72 °C for 20 s. Use the 18S sequence as an internal standard for standardization.