Ominiplant rna kit

Nelumbo nucifera cultivar ‘China Antique’ (named by Prof. Guozheng Huang) was grown in experimental pools in Wuhan, China (30°32′45″N114°24′52″E) by the authors, which is identical with the sequenced one [43 (link)], and has been cultivated for several decades in Wuhan Botanical Garden, Chinese Academy of Sciences. The 16 tissues included leaf, petiole, rhizome (including tip, elongation zone, and internode), root, flower bud, petal, stamen (immature and mature), carpel (immature and mature), receptacle (immature and mature), and seed (seed coat and cotyledon) were collected before 10:00 am in July. For sample harvesting, it is unnecessary to obtain any permission from any authority. All the samples were frozen with liquid nitrogen immediately after harvesting, and then kept in − 80°C freezer until used for RNA extraction.
The RNA reagent (OminiPlant RNA Kit, CWBIO, China) was used to extract the total RNAs. During extraction, genomic DNA was removed with RNase-free DNase I (Thermo, Shanghai, China). The RNAs were reversed with HiScript II One Step RT-PCR Kit (Vazyme, China) to synthesize complementary DNAs (cDNAs) synthesis according to instructions of the Kit.

To analyze the TCP genes expression in different organs of cucumber, we sampled 11 organs of cucumber: root (3 week old seedings), stems (12 week old cucumber plants), leaf (3 week old seedings), cotyledon (3 week old seedings), tendril (12 week old cucumber plants), petal, stamen (1 day before flowering), pistil (1 day before flowering), carpel (1 day before flowering), pericarp (1 day before flowering) and trichome (1 day before flowering). In addition, the expression of 27 CsTCPs in flower buds at different development stages were also examined using real-time quantitative reverse transcription PCR (real-time qRT-PCR). The gene expression pattern analysis in different flower bud development periods was taken from the buds of different lengths, 1, 2, 5 and 10 mm.
Total RNA was extracted using OminiPlant RNA Kit (CWBIO, Beijing, China). The first-strand cDNA was synthesized using HiFiScript cDNA Synthesis Kit (CWBIO). Real-time qRT-PCR with UltraSYBR Mixture (CWBIO) was performed according to the manufacturers’ protocol. CsActin3 (Csa6G484600.1) was used as the internal control. All the qRT-PCR primers were designed using the Genious software according to the cDNA sequences (Tables S1 and S2). The 2^−ΔΔCt method was used to analyze the relative mRNA expression [37 (link)]. Each expression was repeated for three times biologically and technically under identical conditions.

Total RNA of each sample was extracted by OminiPlant RNA Kit (CWBIO, Beijing, China). One microgram (μg) of high-quality total RNA was used for the first-strand cDNA synthesis by TransScript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The qRT-PCR was conducted with three biological replicates, and each biological replicate was technically performed for three times in a 10 μL reaction volume using Hieff qPCR SYBR Green Master Mix (YEASEN, Shanghai, China) on the QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The reaction started with 95 °C for 5 min, then followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. In this study, CsActin (Gene ID: Cs1g05000.1) was used as the internal control, and specific primers of target genes for qRT-PCR were designed by Primer Premier 5 (http://www.premierbiosoft.com/primerdesign/) and listed in Table S3. The relative mRNA expression values were calculated with the Livak method [49 (link)].

Total RNAs were extracted from the salt stress-treated samples using the Omini Plant RNA Kit (CWBIO, Beijing, China). RNA was reverse transcribed into cDNA using a SuperRT cDNA Synthesis Kit (CWBIO, Beijing, China). Then, the cDNA was diluted 1:20 with ddH2O to be used as a template for quantitative real-time PCR (qRT-PCR). Then, qRT-PCR was performed using an AugeGreen™ qPCR Master Mix (US EVERBRIGHT, Suzhou, China) and samples were run in a LightCycler 96 System (Roche, CA, United States) using the following protocol: predenaturation at 95°C for 2 min; 45 cycles of denaturation at 95°C for 15 s and renaturation at 60°C for 60 s; and extension at 72°C for 30 s. The β-actin gene (X79378) was used as an internal reference gene. The primer pairs for qRT-PCR were summarized in Table S5. The relative expression levels were calculated using the 2^-ΔΔCt method, and three biological replicates and three technical replicates were performed for each sample.

The extraction of total RNA from blueberry tissues and cDNA synthesis were performed according to the manufacturer’s instructions of the OminiPlant RNA Kit (CWBIO, China) and HiFiScript cDNA Synthesis Kit (CWBIO, China), respectively. The primers used in the quantitative real-time PCR (qRT-PCR) (Table 1) were designed in Primer Premier 5.0. The qRT-PCR was performed according to the manufacturer’s instructions of the UItraSYBR Mixture (Low Rox) Kit (CWBIO, China).

Blueberry tissue (100 mg FW) from three fruits was ground into powder in liquid nitrogen and transferred into a 1.5-mL RNase-Free tube for total RNA extraction of each treatment and time point.
Total RNA extraction, synthesis of cDNA, and qRT-PCR were performed according to the kit instructions of OminiPlant RNA Kit (CWBIO, China), HiFiScript cDNA Synthesis Kit (CWBIO, China), and UItraSYBR Mixture (Low Rox) Kit (CWBIO, China), respectively. The gene-specific primers used for RT-PCR (VcPLDβ, VcLOX1, and VcCBF) designed using Primer Premier 5.0 are shown in Table 1. The sample of blueberry fruit on the harvest day was used as the mock sample (not shown in this article) following the 2^–ΔΔCt method.