*4.10. Gene Expression Analysis by RT-qPCR*

Total RNA was isolated from gametophores grown in liquid medium, 24h after treatment, with Trizol (Invitrogen, Madrid, Spain) essentially as described in [64]. cDNA was synthesized from 200 ng of total RNA using the iScript™ cDNA Synthesis Kit (Bio Rad Laboratories, Barcelona, Spain). For RT-qPCR, the constitutively expressed *18S* gene was used as a reference gene (Fwd 5 -GGACCGATAGGTCTGGGTAA-3 and Rev 5 - GCAATCCGAAAACTTCACCG-3 ) and for *PpaPrx19* amplification, the primers Fwd 5 - CTCACCACTGACTTCTACGC-3 and Rev 5 -TGGGATGCTGTCCAAGAGTA-3 were used. The PCR reaction contained Bio-Rad 1x iQ SYBR Green Supermix, 0.3 μM primer mix and 2.5 μg of cDNA for a 50 μL final volume. The PCR program comprised a 1 min denaturation step at 94 ◦C followed by 40 cycles of amplification (94 ◦C for 30 s, 58 ◦C for 45 s and 72 ◦C for 1 min) and a final elongation step of 6 min at 72 ◦C. Bio-Rad Optical System Software 3.0 was used for data analysis, and relative expression values were calculated from the resulting Ct values [65].
