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.