Transscript first strand cdna synthesis supermix kit

The qRT-PCR analysis was performed to determine the transcript abundance of VHb and other synthetic genes of the tropane alkaloid biosynthesis pathway, including CYP80F1, H6H, and PMT in the WT, CK, and transgenic hairy roots of H. niger. Total RNA was extracted from H. niger hairy roots using TRIzol A⁺ Reagent (Tiangen Biotech, China). The quality and concentration of RNA were examined by NanoDrop (Thermo, USA) and by visualizing the bands on ethidium bromide-stained agarose gels. The total RNA (1 μg) was reverse-transcribed to obtain cDNA using TransScript First-Strand cDNA Synthesis SuperMix Kit (TaKaRa, Japan), according to the manufacturer’s instructions. qRT-PCR was performed following the instructions from the SYBR-Green PCR Master Mix Kit (Takara, Japan) on a Thermal Cycler Dice Real Time System TP800 (Takara, Japan). An efficiency-corrected comparative Ct method^{49 (link)} was applied and the relative expression of genes was calculated by normalizing the expression of the genes of interest to the abundance of the housekeeping gene (18S ribosomal subunit). All the qRT-PCR experiments were performed in three independent replicates. All the primers used for qRT-PCR are shown in Supplementary Table S1.