Superscripts 2 reverse transcriptase

Seven genes coding for the enzymes in the neoandrographolide biosynthesis were selected for qRT-PCR analysis. Elongation factor 1-alpha was selected as the housekeeping gene. Gene-specific primers targeting 115 to 213 bp regions in the 3′UTR of the transcripts (Supplementary Table 2) were designed using Primer 3 plus software (https://www.primer3plus.com/). First-strand cDNA was synthesized using 1 µg RNA, 0.5 µM oligo-dT primer, and 200 units Superscripts II reverse transcriptase (Invitrogen, USA). The qPCR reactions (10 μl) in triplicate containing 5 μl TB Green Premix Ex Taq II 2X mix (Takara, Japan), 0.5 μM primers, and 15 ng of cDNA were performed in QuantStudio 5 Real-time PCR machine (Thermo Scientific, USA). The qRT-PCR amplification cycle comprised of an initial denaturation at 95 °C for 2 min, 40 cycles of amplification at 95 °C for 30 sec, and 57 °C for 30 sec, a melting curve analysis at 95 °C for 15 sec, 57 °C for 1 min, and 95 °C for 15 sec.

Seven genes coding for the enzymes in the neoandrographolide biosynthesis were selected for RT-PCR analysis. First-strand cDNA was synthesized using 1 µg RNA, 0.5 µM oligo-dT primer, and 200 units Superscripts II reverse transcriptase (Invitrogen, USA). Gene-specific primers for the selected genes were designed using Primer 3 plus software (https://www.primer3plus.com/) to amplify 906 to 2386 bp cDNA fragments (Supplementary Table 1). PCR reactions contained Taq 2X Master Mix RED (Ampliqon, Denmark) to 1X concentration, 0.5 μM primers, and 15 ng cDNA. PCR amplification steps comprised of initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 52–59 °C for 30 sec, and extension at 72 °C for 1 min, final extension at 72 °C for 5 min, and hold at 4 °C.