Ominiplant rna kit dnase 1

Buds of broccoli were frozen in liquid nitrogen. An OminiPlant RNA kit (DNase I) (CWBIO, Beijing, China) was used to extract total RNA from broccoli sprouts, and a HiFiScript cDNA Synthesis kit (CWBIO) was used for qRT-PCR to reverse transcribe the extracted total RNA. Following the UltraSYBR Mixture (Low ROX) (CWBIO) manufacturer’s instructions, qRT-PCR (QuantStudio 5; Thermo Scientific, Waltham, MA, USA) was used to analyze gene expression in the cDNA template (20 µL) reaction. Before sample loading, the cDNA concentration was determined using a spectrophotometer (NanoDrop 2000; Thermo Scientific, Waltham, MA, USA). The gene expression analysis included chlorophyll catabolism genes, carotenoid biosynthesis genes, and flavonoid biosynthesis genes that were associated with color changes. In addition, the gene expression of the relevant transcription factors (the LOB13, Zipper43, NAC92, PIF4, bHLH66, and APL genes) was determined. Primer Premier 5.0 (Premier Biosoft Inc., California, USA) was used to design the primers. The primers used for qRT-PCR are listed in Table S5. Relative gene expression values were normalized to that of the ACT2 gene. The expression of each gene was determined using three biological replicates and calculated using the 2^−ΔΔCT method.

We used an OminiPlant RNA Kit (DNase I) (CWBIO, Beijing, China) to extract total RNA from the fruit samples of C. oleifera according to the manufacturer’s instructions. The concentration and purity of RNA were measured by Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA). RNA integrity was evaluated via agarose gel electrophoresis and Agilent Bioanalyzer 2100 (Agilent, Folsom, CA, USA). About 1 μg of RNA was used to construct cDNA libraries. An Illumina HiSeq2000 platform was used for sequencing in a paired-end mode. The read length was 150 bp. The raw data were deposited to the National Center for Biotechnology Information (NCBI) as project number PRJNA817836.

Total RNA was isolated from non-stressed and stressed leaves of the two hybrids (XY335 and HN138) using the Omini Plant RNA Kit (DNase I) (CWBIO, Beijing, China) based on the manufacturer’s instructions. Total RNA samples were reverse-transcripted to cDNA using the HiFiscript cDNA Synthesis Kit (CWBIO, Beijing, China). We randomly selected fifteen DAPs and designed gene-specific primers for the quantitative real-time polymerase chain reaction assay using Primer Premier 5 Designer software. Real-time PCR was performed on Light Cycler^® 96 using 2X M5 HiPer SYBR Premix EsTaq (Mei5bio, Beijing, China). A steady and constitutively expressed maize gene GAPDH (accession no. X07156) was used as the internal reference gene to normalize gene expression data, together with the forward primer (GAPDH-F: 5′-ACTGTGGATGTCTCGGTTGTTG-3′) and reverse primer (GAPDH-R: 5′-CCTCGGAAGCAGCCTTAATAGC-3′). Each sample had three technical replicates. The relative expression levels were calculated with the 2^−ΔΔCT method [29 (link)].

All expression data of HVA22s under different stresses (blank, drought, salt, high temperature, and low temperature) were acquired from the gene expression database of TM-1(Hu et al., 2019 (link)). The expression profiles of HVA22s from different organs (leaf, stem, root, torus, petal, sepal, calycle, upper stamens, lower stamens, 0–5dpa unseparated ovules, and fibers, 10–25 dpa fiber, 10–20 dpa ovule) were also obtained. The expression of HVA22s was normalized to log₂(FPKM), and heat maps were created with TBtools for visualization.
A 4-week-old salt-tolerant upland cotton variety Han682 was watered with 250 mM NaCl and water and was used as control group. The whole growing process of cotton keeps 16 h light/8 h dark, 25°C. At 0, 3, 6, 12, and 24 h after treatment, root tissue samples were taken for RNA extraction using the OminiPlant RNA Kit (DNase I) (CWBIO), then reverse transcribed into cDNA for qRT-PCR verification of GhHVA22s. Each sample was taken from two cotton seedlings, and three replicates of samples were taken at each point. The NCBI primer design website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design primers for GhHVA22s. GhUBQ14 was used as internal control to standardize the expression of target genes. Gene expression was computed by 2^-ΔΔCt, and Ct was the cycling threshold. The primers are listed in Table S7.

In order to obtain the genomic sequences of the FhTPS genes, DNA was extracted from flowers of Red River^® using the NuClean Plant Genomic DNA Kit (CWBIO) according to the manufacturer’s instructions. RNA was extracted from samples using the OminiPlant RNA Kit (DNase I) (CWBIO) following the manufacturer’s standard protocol. The purity and concentration of RNA were assessed using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). cDNA was synthesized in a final reaction volume of 25 µl from total RNA (1 μg) using Oligo d(T)15 primers together with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s specifications.

Three developmental stages of the flower of M. candidum including closed buds with white petals (McI), closed buds with pink petals (McII), and opened buds with pink petals (McIII) were collected and immediately frozen in liquid nitrogen. Three replicates of each stage sample were taken from three seedlings. Total RNA was extracted by using an OminiPlant RNA Kit (DNase I) (CW2598, CWBIO, Taizhou, China) according to the manufacturer’s protocol. An Agilent 2100 Bioanalyzer was utilized to assess the quality of the RNA (Agilent Technologies, Palo Alto, CA, USA). To generate a cDNA library, we fragmented the mRNA using a fragmentation buffer and reverse-transcribed the resulting small fragments into cDNA using random primers. The second strand cDNA was synthesized by employing DNA polymerase I, RNaseH, dNTP, and buffer. After purification with the poly(A) and PCR extraction kit (Qiagen), the synthesized products were further purified using the QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands) and then ligated to Illumina sequencing adapters. Afterward, the second-strand cDNA was digestedusing the enzyme UNG (Uracil-N-Glycosylase), and the resulting products were separated by size on an agarose gel before being amplified by PCR. The PCR products were then sequenced on Illumina HiSeq TM 4000 platforms.

Total RNA was extracted from seed tissues from control and PEG-treated germinating seeds (for both wild-type Vp16 and mutant vp16) using Omini Plant RNA Kit (DNase I) (CWBIO, Beijing, China). Using HiFiscript cDNA Synthesis Kit (CWBIO, Beijing, China), the extracted RNA was then reverse-transcribed in a 20 µL total volume as per manufacturer’ guidelines. Gene-specific primers for twenty-eight randomly selected DAPs (Table S9) were designed for qRT-PCR analysis using Primer Premier 5 software. The expression of these genes was studied by qRT-PCR in a C1000 (CFX96 Real-Time System) Thermal Cycler (Bio-Rad) using 2× Fast Super EvaGreen ^® qPCR Mastermix (US Everbright Inc., city, CA, USA); GAPDH was used for housekeeping. Each 1 µL of cDNA template was mixed with 1 µL of each primer (50 pmol), 10 µL of 2× Fast Super EvaGreen ^® qPCR Mastermix (US Everbright Inc., city, CA, USA) and 7 µL ddH₂O in a 20 µL reaction mixture. The PCR schedule was run as follows: 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s and 55 °C for 30 s. The relative expression level of each gene was determined using Livak and Schmittgen’s cycle threshold (2^–ΔΔCT) method [90 (link)], with three technical replications for each sample.