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).