C1000 touch thermal cycler system

Nine candidate unigenes involved in anthocyanin synthesis from three roselle cultivars were screened for qRT-PCR analysis. First, purified RNA was reverse-transcribed to first-strand cDNA using a cDNA Reverse Transcription Kit (TransScript One-step gDNA Removal and cDNA Synthesis SuperMix) [56 (link)] based on the manufacturer’s instructions. Primers for the candidate unigenes were designed using IDT (https://sq.idtdna.com/Primerquest/Home/Index) (accessed on 10 January 2022) and Primer3 (Primer3 Input). Before qRT-PCR, the presence of primer dimers and primer quality were determined using standard PCR. The specific steps of qRT-PCR were conducted using PerfectStart^TM Green qPCR SuperMix as per the manufacturer’s instructions on a C1000 Touch™ Thermal Cycler System (Bio-RAD). Three biological replicates were performed for each sampling period, and three technical replicates were performed for each qRT-PCR to ensure the reliability of experimental data. The relative expression levels of differential genes were calculated using the 2^−ΔΔ Cp method, and the housekeeping gene Actin7 was used as an internal reference.

DEGs from the PCP biosynthesis pathway were selected for further testing by qRT-PCR. Total RNA was isolated from two germplasm rhizomes with the largest PCP content contrast and reverse transcribed to cDNA with HiScript^® II Q RT Super Mix (+gDNA wiper) (Vazyme Biotech, Nanjing, China). We used a C1000 Touch™ Thermal Cycler system (Bio-Rad, Hercules, CA, USA) and SYBR Premix Ex Taq to perform qRT-PCR. We selected a P. cyrtonema ACTIN to serve as the inner reference. All primers are listed in Table S7. The 2^−ΔΔCt method was used to calculate gene expression. Three biological replicates were used for each sample.

Total RNA (50–100 mg) was isolated from root and shoot of B. distachyon using RNAzol^®RT (MRC, Cincinnati, OH, USA) following manufacturer’s instruction. RNA sample were quantified using NanoDrop 2000 (Thermo Scientific Inc., Waltham, MA, USA) and stored at −80 °C till further use. Total RNA (1.0 μg) was converted to ss-cDNA using Transcript one-step gDNA removal and cDNA synthesis Super mix (Transgen, Beijing, China) as per manufacturer’s protocol. qRT-PCR reactions were performed in BIO-RAD C1000 Touch™ Thermal cycler system using iTaq™ universal SYBR^® green Supermix qPCR Kit (BIO-RAD, Hercules, CA, USA). Total cDNA was diluted to ∼25 ng/μL and a total 100 ng was used in a 20 μL reaction mixture. For each reaction, three technical replicates were used along with no template control to check for contaminants. The following thermal cycling programme was used for all qRT-PCR reactions: 3 min at 95 °C, 3 s at 95 °C and 45 s at 60 °C for 40 cycles, which includes data acquisition. Finally, a dissociation curve analysis was performed from 65 to 95 °C in increments of 0.5 °C, each lasting for 5 s, to confirm the presence of a specific product. Concentration of ACT2 (At3g18780) was used to normalize the gene expression in different samples. Log fold change in expression values were calculated using the 2^−ΔΔCT method (Livak & Schmittgen, 2001 (link)).