Cfx connect real time pcr detection system

Total RNA was isolated from various tissues of the WT plants, including the root, stem, leaf, flag leaf, tiller, and panicle, and WT developing seeds (3, 6, 9, 12, 15, 18, and 21 days after pollination), and WT embryo and endosperm of the dry seeds, and 6 days embryos after pollination of the WT and transgenic rice lines using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. Developing seeds were collected at 6 days after pollination (DAP), and the embryos were isolated under a microscope using a sterile blade and immediately placed in liquid nitrogen, following which they were stored at −80°C until analysis. The RNA samples were reverse-transcribed into first-strand cDNA using a PrimeScript RT Reagent Kit (Takara, Japan) and qRT-PCR reactions were carried out on a Bio-Rad Laboratories CFX Connect^TM Real-Time PCR Detection System with SYBR Premix Ex Taq (Takara, Japan). All experiments were conducted with at least three biological replicates. The qRT-PCR reactions were normalized based on the rice Actin1 gene (OsActin1), which was used as an internal control using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). The primers designed by Primer Premier 5.0 software for the analysis of transcript levels are listed in Supplementary Table S1.

The total RNA for qRT-PCR was isolated from the collected roots of each biological replicate by using an RNeasy Kit (Invitrogen) following the manufacturer’s instructions. DNA impurities in the isolated RNA were digested before synthesizing the cDNA by adding gDNA Eraser (TAKARA, Dalian, China) and incubated for 2 min at 42 °C. Then, the first-strand cDNA was synthesized from 1 μg of total RNA with the Prime Script™ RT Reagent Kit (TAKARA, Dalian, China), following the manufacturer’s instructions. Quantitative PCR reactions were performed with a Bio-Rad system (CFX Connect TM Real Time PCR Detection System, CA, USA) and TAKARA SYBR Green Master mix (TAKARA, Dalian, China) in a 10 µL reaction volume. A single 10 μL PCR included 5 μL of TAKARA SYBR Green Master mix (TAKARA, Dalian), 1 μL of cDNA template, and 10 μM each of forward and reverse primers (sequences used are described in Table S1). The actin gene GmActin11 was used as an internal control [78 (link)]. The PCR cycling parameters were as follows: pre-incubation at 95 °C for 2 min, 39 cycles of 95 °C for 5 s, 60 °C for 30 s, and 95 °C for 5 s. PCR reactions for each of three biological replicates were performed in technical triplicate. The relative expression level was normalized with the 2^−ΔΔCt method [79 (link)].

Total RNA from muscle tissue samples was prepared as described in Section “Clustering, Sequencing, and Transcriptome Assembly.” The samples were diluted to 1 μg/ml with DEPC-treated double distilled water and first strand cDNA was prepared using the PrimeScript^TM first strand cDNA Synthesis Kit (TaKaRa). We selected five DElncRNAs and their targeted DEGs; the lncRNAs (CREB5, ITGB3, TCONS_00007120, IGF2, TCONS_00024310, RTL1, XR_314844.1, PAX7, TCONS_00034044, MEF2C, TCONS_00027657, TCONS_00014143, TCONS_00027958, and XR_001351404.1) were involved in the process of muscle growth. The relative expression of 15 candidate differentially expressed genes/lncRNAs in the nine muscle tissue samples was analyzed by qRT-PCR the using the cDNA as a template and SYBR^®Premix Ex Taq^TM III Reagent Gold (TaKaRa) on the CFX Connect^TM Real Time PCR Detection System. Details of the primers designed to amplify the 15 candidate differentially expressed genes/lncRNAs are shown in Supplementary Table S1; the β-actin gene as the internal reference. The mean number of cycles required to pass the fluorescence threshold (Ct value) of each sample was used to calculate relative gene expression using the 2^–ΔΔCt method. The reliability of the sequencing results was evaluated by correlation analysis of the RT-qPCR and RNA-Seq data.