2.8 RNA extraction
In order to acquire total RNA extraction, shoot samples were powdered in
the liquid nitrogen (100 mg) using 1000 µl Trizol reagent
(Sigma-Aldrich). Then, 200 µl of chloroform was added into the solution
and after 15 minutes of incubation at room temperature, the samples were
centrifuged at 12,000 g for 10 minutes at 4 °C. The RNA was precipitated
in the aqueous phase using propanol and saturated salt solution (0.4 M
sodium citrate and 1.2 M sodium chloride) and thereafter washed with
70% ethanol. The resulting samples were carefully air-dried and finally
air-dried RNAs were dissolved using DEPC (CinnaGen) water and stored at
-80 °C until cDNA synthesis. After extracting, the RNA quantity was
determined by detection of its absorption ratio at 280 and 260 nm (the
acceptable absorption ratio for these two wavelengths is 1.5 to 2.1)
using picodrop (Table S2). RNA quality was investigated by its loading
in agarose gel (Razeghi and Leister, 2013) (Figure 1A).