RT-qPCR Analysis of Gene Expression

Total RNA was extracted from in vitro samples using the RNeasy^® Plant Micro and RNeasy^® Plant Mini kits (Qiagen) according to the manufacturer’s instruction. cDNAs were obtained from 2 µg of RNA using the Superscript™ II reverse transcriptase (Invitrogen) according to Solís et al. (2012) (link). RT-qPCR analyses were performed using the SsoAdvanced™ Universal SYBR^®Green Supermix on the iQ™5 Real-Time PCR Detection Sytem (Biorad). The oligonucleotides used are described in Supplementary Table S2, and qPCR conditions were as previously described (Berenguer et al., 2017 (link)). All qPCRs were run in duplicate, and the Cyclophilin gene was used as the internal reference gene. Transcript levels were normalized to the vacuolated microspore levels. Data were analysed with the Bio-Rad CFX Manager 3.0 (3.01224.1015) (Biorad), using the Livak calculation method (Livak and Schmittgen, 2001 (link)).