Trizol rna purification kit

The sequences of the genes used for qRT-PCR validation were provided (details listed in Supplementary Data Sheet S1). Gene-specific primers (Supplementary Table S1) were designed using Primer3² Total RNAs were extracted from leaves and from sepals, petals, stamens, and pistils at 8–10 mm flower buds development stage using TRIzol RNA purification kit (TaKaRa, China). One microgram of total RNA was used in reverse transcription in a total reaction volume of 20 μL in the presence of 6-mer random primers and an oligo primer according to the protocol provided by manufacturer TaKaRa (Yan et al., 2011 (link)). The standard curve for each gene was obtained by real-time PCR with five dilutions of cDNA. The reactions were performed in 20 μL volumes each containing 10 μL 2× SYBR Green Mastermix (TaKaRa), 300 nM of each primer and 2 μL of 10-fold diluted cDNA template (Yan et al., 2014 (link)). The PCR reactions were run in a Bio-Rad Sequence Detection System. Three biological replicates were performed for each analysis. RhGAPDH (AB370120) was used as control. Quantification of gene relative expression in different organs was performed using the delta-delta Ct method as described by Livak and Schmittgen (2001) (link). All data were expressed as the mean ± standard deviation (SD) after normalization.

Genome DNA and floral transcriptomic RNA were isolated from Hybrid Tea Rose ‘Samantha’ leaves for paired-end sequencing on the Illumina 150 × 2 platform. ‘Samantha’ produces scented, medium-sized red flowers and dark green, leathery foliage, for which it is cultured worldwide [15 (link)]. Genomic DNA was isolated from young leaves of rose plants as described by Aldrich and Cullis (1993) [16 (link)], but with 1% (w/v) polyvinylpyrrolidone-10 added to the DNA extraction buffer. Total RNA was extracted from rose floral organs using a TRIzol RNA Purification kit (TaKaRa, China) following the manufacturer’s instructions. The RNA was treated with RNase-free DNase (Tiangen, China) to remove residual genomic DNA. First-strand cDNA synthesis was conducted using 20-μL reaction mixtures containing 1 μg of total RNA and oligo(dT) primers (TaKaRa, China).

Total RNA samples were extracted from the tibial GP using TRIzol RNA purification kit (Takara, Japan). After extraction of total RNA samples from tibial GP tissue, the samples were used for PCR and RT-qPCR. Transcripts from each sample were amplified in triplicate and detected using an SYBR Green PCR Master Mix (Applied Biosystems). Premier 5.0 software was used to design PCR primers based on the sequences of the HDCA1, MTA1, H4, and PCNA and housekeeping 18s rRNA reference gene in the NCBI GenBank. The primer sequences are shown in Table 1. The primers were synthesized by shanghai Generay Biotech Co.,Ltd. Operate according to the instruction of SYBR® Premix Ex Taq II (Takara, Dalian), and use QuantStudio 6 system (ABI, USA) for PCR reaction.