Hiscript 2 q select rt supermix for qrt pcr

Nuclear and cytoplasmic RNA extraction was performed using the PARIS™ Kit (Invitrogen), and the total RNA was isolated from the cells and skin using the RNAsimple total RNA Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. For mRNA verification, total RNA (1 μg) was reverse transcribed using the HiScript II Q Select RT SuperMix for qRT-PCR (Vazyme, Nanjing, China), and cDNA (10 ng) was used for the quantitative mRNA analysis, which was performed using AceQ qPCR SYBR^® Green Master Mix (Vazyme, Nanjing, China). For lncRNA verification, the lnRcute lncRNA First-Strand cDNA Kit (Tiangen, Beijing, China) was used for cDNA synthesis, and the lnRcute lncRNA qPCR Kit (SYBR Green, Tiangen, Beijing, China) was used for qRT-PCR. The data were analyzed using QuantStudio^® 5 (Applied Biosystems, Massachusetts, USA), according to the manufacturer’s instructions. The relative expression levels were calculated using the 2^−ΔΔCt method [28 (link)] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. U6 RNA served as a positive control for the nuclear gene expression. The specific primers are listed in Table S4.

The total RNA of plant materials was extracted using Trizol reagent (Coolaber, Beijing, China). One microgram of total RNA was used to synthesize cDNA with HiScript II Q Select RT SuperMix for qRT-PCR (Vazyme, Nanjing, China). qRT-PCR was performed on a CFX384 real-time system (BIO-RAD, Hercules, CA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The experiments were repeated three times independently. The actin2 gene was used as an internal control. The primers used in this assay are listed in Table S1.

Total RNA was extracted from the aforementioned plant material using Trizol reagent (Coolaber, Beijing, China). Moreover, 1 μg of total RNA was used to synthesize cDNA using HiScript II Q Select RT SuperMix for qRT-PCR (Vazyme, Nanjing, China). The qRT-PCR assays were subsequently performed using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with a reaction volume of 10 μL. The qRT-PCR master mix used was ChamQ universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Three biological replicate samples were taken for sunflower RNA extraction, and these experiments were independently repeated three times. The HaTublin gene was used as a reference gene. The primers used for qPCR are listed in Table S3.