Takara minibest plant rna extraction kit

Gene expression level in A. flavus mycelial samples was measured by qRT-PCR. There were three biological replicates per sample. Total RNA was extracted from frozen mycelial samples that had grown for two, three, four, or five days at 28^°C and 37^°C, using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 500 ng of total RNA per sample using a HiScript-II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Biotech Co., China) following the manufacturer’s instructions. qRT-PCR was then performed using ChamQTM SYBR®qPCR Master Mix (Vazyme Biotech Co., China) in a CFX connect system (Bio-Rad, United States) and primers listed in Supplementary Table S1. The specificity of amplification was confirmed based on the melting curve. Relative gene expression was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) with β-tubulin as the internal control.

Total RNA was extracted from mature-stage fruits, using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan), following the manufacturer’s instructions. cDNA was synthesized using a TransScript^® Reverse Transcriptase Kit (TransGen Biotech, Beijing, China) for real-time RT-PCR (TaKaRa, Japan) following the manufacturer’s protocol. Specific primers were designed to amplify the ORF based on the complete sequence of coding sequence (cds) from the Prunus persica (http://www.ncbi.nlm.nih.gov; accession umber ABV32551.1); primers were synthesized by BGI (Beijing, China). Forward and reverse primer sequences were: ChSPS1-F (5′-ATGGCGAGCAACGATTGGATA-3′) and ChSPS1-R (5′-CTACGTCTTGACAACTCCGA-3′) respectively. PCR was carried out as follows: initial denaturation at 94°C for 1 min; followed by 36 cycles of denaturation at 94°C for 30 s, annealing 59.2°C for 30 s, and elongation at 72°C for 1 min, with a final 10min extension step at 72°C. PCR products were analyzed by electrophoresis on 1% (w/v) agarose gels. PCR products were ligated into the pMD18-T cloning vector (TaKaRa, Japan), and the pMD18-ChSPS1 was then transformed into E. coli DH5α competent cells (TransGen Biotech, Beijing, China) for sequencing by BGI.

RNA was extracted from a leaf sample using the TaKaRa MiniBEST Plant RNA Extraction Kit (Code 9679) (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China) in accordance with the manufacturer’s instructions. Following the same instructions from the TaKaRa PrimeScript™ II 1st Strand cDNA Synthesis Kit (Code 6210) (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China), first-strand cDNA was synthesized by reverse transcription reaction using Oligo dT Primer and Random 6 mers as primers, PrimeScript II RTase as reverse transcriptase. The quality and quantity of the extracted nucleic acid were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA was stored at −80 °C, and the cDNA was stored at −20 °C until further use. The product was subsequently employed as a template for RPA.

Total RNA from each sample was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). The RNA integrity value (RIN) was checked with a Bioanalyzer 2100 (Agilent Technologies Co., Ltd., Palo Alto, CA, USA). The total RNA quality was assessed by means of 1.2% agarose gel electrophoresis. The RNA samples with RIN > 8 were used for subsequent analyses.
For the Iso-Seq analysis, RNA from the Y and R samples were extracted separately and then mixed equally into one sample (designated R1). Similarly, the W and S extracts were mixed to form the S1 sample. The Iso-Seq library was prepared in accordance with the isoform sequencing protocol using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol, as prescribed by Pacific Biosciences (PN 100-092-800-03), on a PacBio platform by Novogene Co., Ltd. (Beijing, China).
Total RNA from the taproots of the four materials was extracted for each sample and used as the input material to create sequencing libraries using the NEBNext Ultra^TM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) [58 ], following the manufacturer’s instructions, and deep sequenced by Novogene Co., Ltd. (Beijing, China) using an Illumina sequencing platform.

Spikelets and seeds were sampled from the fon7 mutant plants and the T-DNA-tagged line as well as from the corresponding wild-types (Ilpum and Dongjin, respectively) in three biological replicates, with each replicate containing three technical repeats. Total RNA was extracted from the harvested plant samples using the TakaRa MiniBEST Plant RNA Extraction Kit (TaKaRa Bio, Kusatsu, Japan), and first-strand cDNA synthesis was carried out using oligo (dT) primers and M-MLV reverse transcriptase (Promega, Madison, WI, USA). Quantitative real-time PCR (qRT-PCR) was performed using sequence-specific primers and SYBR Premix ExTaq (TaKaRa, Japan) on the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Expression levels of FON7 were normalized relative to those of Actin, a housekeeping gene, and relative gene expression levels were calculated using the ΔΔCt method.

Total RNA was isolated from seedling leaves using TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, China). About 0.5 μg total RNA was reverse-transcribed to the first-strand cDNAs using PrimeScript TM RT Master Mix (TaKaRa, China). Real-time quantitative PCR (qRT-PCR) detection was performed on a CFX96 Real-Time PCR Detection System using SYBR® Premix Ex Taq TM II (TaKaRa, China). The assaying genes included the upstream-downstream genes of OsXDH such as UO (LOC4324793), ALN (LOC4337428), and AAH (LOC4341777), and leaves senescence related genes such as SGR (LOC4347672), WRKY23 ((LOC4324161), WRKY53 (LOC4338474), GH27 (LOC4323975), Pse (t) (LOC4337812), ACD1 (LOC4331611), and XERO1 (LOC4350453). Ubi2 (LOC4332169) was used as the reference gene. Three plants were selected for each treatment group in each replicate experiment. During reverse transcription PCR (RT-PCR), OsXDH transcripts were co-amplified with OsACTIN (LOC4333919) mRNA as an internal control. All the primers used to gene expression analysis above were listed in Supplemental Table 2.

Total RNA was extracted from 5 g samples of roots, stems, leaves and seeds, respectively, with the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa). First-strand cDNA was synthesized and qPCR was carried out using a LightCycler^®480 System Real-Time PCR as described by Feng et al. (2019) (link). All primers for the target genes are listed in Table 1. Relative expression of target genes was evaluated by the 2^–ΔΔCt method (Li et al., 2018 (link)). The experiments were conducted in three biological replications.