4.6 RNA extraction and qRT-PCR
For qRT-PCR analyses, total RNA was extracted using Tri reagent
(Sigma-Aldrich) according to the manufacturer’s instructions, and 3μg of
RNA was converted to first strand cDNA using reverse transcriptase
(Promega, United States) and oligo-d(T) primers according to the
manufacturer’s instructions. qRT-PCR was performed according to the
Power SYBR Green Master Mix protocol (Life Technologies, Thermo Fisher,
United States), using a Rotor-Gene Q machine (Qiagen). Supplemental
Table 1 lists the specific primers used in this study. The housekeeping
gene coding for ribosomal protein RPL8 (accession number
Solyc10g006580) was used for the normalization of gene expression in all
qRT-PCR analyses. Relative expression quantification was calculated
using copy number method for gene expression experiments (D’haene et
al., 2010).