2.5 RNA extraction and quantitative Real-time PCR (qRT-PCR)
RNA from anthers was extracted using the total RNA pure kit (Aidlab Biotech, Beijing, China) as per the manufacturer’s protocol. The concentration of RNA in each sample was determined using a NanoDrop (Denovix, Spectrophotometer, USA). RNA was stored at -80 ˚C until used. Aerosol protected pipette tips were used throughout the extraction and quantification steps to prevent RNA contamination. The first-strand cDNA was synthesized by using 2000 ng µl-1 purified total RNA, RT/RI enzyme and oligo (dT)18 primer (TransGen Biotech, Beijing, China) following the instructions of kit. The qRT-PCR was done using Top Green qPCR SuperMix in a volume of 20 µl according to the manufacturer’s instructions and applied to the QuantStudio 5 real-time PCR system (Applied Biosystems, Beijing, China). The amplification of the wheat actin was used as an internal control for normalizing all data.Actin gene were used as a negative control in this experiment. All genes were run on a 96-well QuantStudio 5 (Applied Biosystems, Beijing, China) with the following conditions: pre-denaturation at 95 °C for 10 min, 40 cycles (95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s) were performed. The 2 −ΔΔCt method (Pfaffl, 2001) was used to evaluate the presence of genes with three biological replicates and four technical replicates, which prove the timeline of anther development.