Primescript rt kit with gdna eraser

The total RNA from the pomegranate seeds was extracted using the cetyltrimethyl ammonium bromide (CTAB) method (Solarbio, Beijing, China). The cDNA was generated using one microgram of RNA by using a PrimeScript^TM RT Kit with gDNA Eraser (TaKaRa, Dalian, China) following the manufacturer’s protocol. A similar procedure was followed during the extraction of total RNA and cDNA from the transgenic lines of Arabidopsis.

Cell RNAs were converted to cDNA using a PrimeScriptTM RT Kit with gDNA Eraser (Takara, Dalian, China) for the mRNA and pri-miRNA qPCR. The cDNA used for qPCR of miRNAs was synthesized using a 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The qPCR was performed using TB Green™ Premix Ex TaqTM II (Takara, Dalian, China) on a Bio-Rad CFX96 Touch™ Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The procedure of qPCR used in quantifying mRNA and pri-miRNA was as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s, and the expression abundance was normalized relative to that of GAPDH. The reactions of miRNA qPCR were incubated at 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s. Primers used in qPCR were designed by oligo 6. The sequences of pri-miRNAs were obtained from the Ensembl database by extending the 5′ flanking and 3′ flanking sequences to 150 bp of the selected miRNAs. For miRNA qPCR detection, the mature sequence of miRNAs was used as the forward primer, and the reverse universal primer was provided by the miRNA cDNA Synthesis kit. U6 was used as the internal control in quantifying miRNAs. All the primers are listed in the Supplementary Materials, Table S1.

Four-week-old plants were treated with 200 μM CdCl₂ for 6 h and the roots were harvested for total RNA analysis. Total RNA was extracted with TRIzol (Invitrogen, United States), precipitated with an equal volume of isopropanol, washed with 75% ethanol, and dissolved with RNase-free water. The cDNA templates were synthesized using the PrimeScript^TM RT Kit with gDNA Eraser (Perfect Real Time) (TAKARA, Japan) following the manufacturer’s protocol. The relative expression of genes in roots was determined by quantitative RT-PCR performed in an Applied Biosystems StepOne^TM Real-Time PCR System using SYBR Premix Ex-Taq (TAKARA) according to the manufacturer’s protocol. Primers used in the assays are listed in Supplementary Table S1. The expression data were normalized to Actin2 or sand.

RT-qPCR was used to verify the reliability of transcriptome results and the final candidate genes. We took a 1 ug RNA sample from the RNA-Seq for RT-PCR. We used a PrimeScript^TM RT kit with gDNA Eraser (Takara, Dalian, China) for cDNA reverse transcription according to the instructions. The primers were designed using Primer 5 software with actin as the internal reference gene. SYBR^® Premix Ex TaqTM II (TliRNaseH Plus) was used to complete all RT-PCR analyses on the fluorescence quantitative PCR instrument (ABI7500, Applied Biosystems, Foster City, CA, USA). Each parent had three biological replicates and each biological replicate had three technical replicates; the relative expression levels of target genes were calculated by the 2^−ΔΔCt method.

RNAprep pure plant kit (Biofit, Chengdu, China) was used to isolate total RNA from fresh Wanhu No.5 and Wanhu No.6 flowers (100 mg). According to the manufacturer’s instructions, RNA (1 μg) was used to synthesize cDNA using PrimeScript^TM RT kit with gDNA eraser (January, Perfect Real Time, Takara, Tokyo, Japan). Using QuantStudio 6 Flex real-time PCR system (Thermo Fisher, Waltham, MA, United States) and SYBR^® PremixExTaq^TMII (2x) (Japan, Takara), the gene expression level was detected by qRT-PCR. We used NCBI-BLAST online software² to design fluorescent quantitative primers for the key genes in the terpene synthesis pathway (Supplementary Table 1). The results are attached in Figure 11. The reaction steps are 50°C 2 min, 95°C 30 s, 95°C 5 s, 60°C 34 s, 40 cycles, and 72°C for 10 min (Jin et al., 2013 (link)). The 2^–ΔΔCT method was used to calculate the relative gene expression, and the experiment was repeated three times (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from different samples using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using a PrimeScript^TM RT kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative reverse transcription PCR (RT-qPCR) analysis was performed using the CFX96 real-time system (Bio-Rad, Singapore) with a SYBR Premix ExTaq^TM Kit (TaKaRa), as described previously [29 (link)]. The primers used in the RT-qPCR are shown in Table S2. The 2^−ΔΔCt method was adopted to calculate the relative expression levels. In this study, B. mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was used as the reference gene. The statistical significance between treatments was analyzed using SPSS Statistics software (V 26.0. IBM, NY, USA). Three biological replicates were used.

Total RNA was isolated from leaves and roots using the Plant Total RNA Extraction Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the kit instructions. RNA concentration and mass were evaluated by detection of the A260/A280 and A260/A230 ratios, respectively, using a spectrophotometer (NanoVueTM plus, Wilmington, DE, USA). Genomic DNA contamination removal and first strand cDNA synthesis were performed using the PrimeScriptTM RT kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions. According to the standard that 10 μl reaction system can use up to 500 ng of total RNA, the RNA volume is calculated to ensure that all the added RNA is reverse transcribed into cDNA. Finally, we will dilute the cDNA concentration of all samples to 50 ng/μl according to the method described. The qPCR reaction conditions were as follows: 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C, followed by 65–95°C melting curve detection. The negative reverse transcriptase reaction (no reverse transcriptase) was carried out after DNA contamination was removed by gDNA eraser. The CT value obtained by fluorescence quantitative PCR was compared with that of the negative control.