Primescript rt reagent qpcr kit

Fifteen differentially expressed transcripts were selected for validation of sequencing data using qRT-PCR. Specific primers were designed by Primer 5 software (Premier Biosoft International, Palo Alto, CA). Total RNA was extracted using an RNAprep pure Plant Kit (Tiangen, Beijing, China) from the styles. One microgram of total RNA was used to synthesize cDNA using a PrimeScript® 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Then, qRT-PCR was performed on the ABI 7500 Real-Time PCR System (Applied Biosystems) using a PrimeScript™ RT reagent qPCR Kit (Takara, Dalian, China). A GADPH (GE651107) gene was utilized as the reference gene. The real time PCR program was as follows: 95 °C for 15 s, 40 cycles at 95 °C for 5 s, 60 °C for 34 s. Each reaction was repeated three times for biological and technical replicates, respectively. The relative quantitation of the gene expression was calculated using the 2^−ΔΔCt method [64 (link)].

One microgram of total RNA of each sample, including seven different developmental stages of embryo and six different tissue types, were used for first-strand cDNA synthesis with a PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The primers of CiCHS2 and CiCHS3 came from our laboratory previously [10 (link)]. Gene-specific primers of CiCHS1 were designed using Primer 5.0 (Table S1). Expression profiles of CiCHSs were detected using qRT-PCR on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) and were normalized to the 18s RNA housekeeping gene [45 (link)]. The qRT-PCR reactions were performed according to the PrimeScript RT reagent qPCR Kit manual (TaKaRa, Dalian, China). The 20 μL reaction volume contained 10 μL of 2 × SBYR Premix Ex Taq II, 0.4 μL of 50 × ROX reference Dye, 0.4 μL of each primer (10 μM) and 80 ng of cDNA. The PCR program was 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 61 °C for 34 s; and a dissociation stage. The relative expression of the CiCHSs was calculated using the 2^−ΔΔCt method. Each reaction was performed in 3 biological replicates. The expression patterns of CiCHS2 and CiCHS3 at four time points (August 29, September 12, September 26, and October 10) of “YLC28” were analyzed in our previous study [10 (link)].

Total RNA extracted using the RNeasy Plus Mini Kit was treated with TURBO DNase to remove genomic DNA. First-strand cDNA was synthesized from 1 μg DNA-free RNA in a reverse-transcription reaction using random hexamer primers and the MultiScribe reverse transcriptase from a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The cDNA samples were diluted 10-fold in nuclease-free water and used as templates for qRT-PCR analysis. The qRT-PCR primer pairs are listed in Additional file 6. All of the analyzed unigenes were examined using three technical replicates and three biological replicates for each stage. The qRT-PCR was conducted using an ABI 7500 Real-Time PCR System (Applied Biosystems), SYBR Green, and a PrimeScript™ RT Reagent qPCR Kit (Takara, Dalian, China). Relative transcript abundances were calculated according to the comparative cycle threshold method, with GAPDH as an internal standard [68 (link)]. Pearson correlation analyses were completed using the SPSS Statistics 17.0 software package (SPSS Inc., Chicago, IL, USA). Correlation analyses were conducted using the log ratios (i.e., log₂ fold-change between stages) of unigene expression levels determined by qRT-PCR and RNA-seq.