Omniscript rt kit protocol

Three plants of each transgenic line and controls from 3-month-old seedlings in the greenhouse were selected randomly, and the fully expanded upper leaves of the seedling were collected, immediately put into liquid nitrogen, and transported to the laboratory. Total RNA was extracted from 100 mg of leaves using the RNeasy extraction kit (Qiagen, Germany). cDNA was synthesized using 5 μl of total RNA (500 ng) according to the Omniscript RT kit protocol (Qiagen) as described by the manufacturer and it was subsequently used as target for the qPCR reaction. According to the full sequence data of the targeted genes, fluorescence quantitative PCR primers were designed as shown in Table 1. With the previous reverse transcript cDNA as the template, 2 × Sybr Green qPCR Mix was used for fluorescence quantitative PCR. According to the cycle amounts that the fluorescent signal of each PCR reaction tube reaches in the designed threshold (CT value) and the standard curve developed with the use of standards that have a known expression amount, the abundance of the each Bt gene in the synthesized cDNA, with mRNA as transcription, was calculated from the standard curve [13 ].

Cultured cells were collected, and RNA was prepared using an RNA kit according to the manufacturer’s instructions (Transgen, Beijing, China). cDNA was synthesized using 6 µl of total RNA (300 ng) according to the Omniscript RT kit protocol (Qiagen, Germany) as described by the manufacturer. Commercial primers for Syk, p65, and β-actin (DHS57991, DHS445896, DHS938729) were purchased from XY biotech (Shanghai, China). Real-time polymerase chain reaction (PCR) was performed following the instructions of the top green quantitative PCR SuperMix (Transgen) in a real-time PCR machine (MYGO PRO, IT-IS, Ireland). Expression of target genes was corrected by the expression of β-actin housekeeping gene and calculated using the 2^−ΔΔCt method.