Sybr green qrt pcr kit

Approximately, 0.1 g of the immature ear tissue (1.5–2 mm) was collected from each of the recombinant lines, parent lines and 105 inbred lines. Tissue samples of seedling roots, seedling leaves, internodes, mature leaves, 1-mm ears, 4-mm ears, and 2- and 4-cm tassels from B73, and SPMs, SMs, and floral meristems (FMs) from NIL^qknr6 and NIL^qKNR6 were collected for the extraction of total RNAs using the Pure Link Plant RNA Reagent (Ambion, Austin, TX, USA). Approximately, 1.0 μg of RNAs were reversely transcribed by the M-MLV reverse transcriptase (Life Technologies, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green qRT-PCR Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions with three biological replicates, each with three technical replicates. The maize ACTIN (Zm00001d010159) was used as the internal control. All reactions were carried out on the CFX96 Real-time system (Bio-Rad, Hercules, CA, USA). The relative expression of the gene was calculated by the 2−ΔΔCt method.

Total RNA of tissue and adipocytes were extracted with Trizol (Invitrogen, CA, USA). Reverse transcription of total RNA (1 μg) was performed with a high-capacity cDNA reverse transcription kit (Takara, Shiga, Japan). Amplification reactions were conducted using a SYBR Green qRT-PCR Kit (Bio-Rad Laboratories, CA, USA) with the Bio-RAD CFX 96 PCR system [25 (link)]. The amplification of each gene was verified by melting points. All primer sequences are listed in Table S1.