High quality RNA isolation and exogenous reference gene for real-time PCR in Cedrela odorata seed physiology studies

Seed banks represent an important strategy for the conservation of forest genetic resources, although a basic understanding of the physiological changes that seeds undergo during storage that affect quality and germination is still lacking for most tropical and subtropical species. Here, we describe the optimisation of an RNA isolation procedure and reference gene normalisation for expression analysis in Cedrela odorata (cedro or Spanish cedar) seeds during different physiological states, as well as in the steady-state stem and leaf. The expression profiles of five endogenous candidate reference genes (18S, EF1α, GAPDH, CDC27B, PP2A2) and an exogenous (HMBS) gene were evaluated by using dedicated algorithms, including Genorm, Normfinder, Bestkeeper and ΔCt. We found that the expression of all endogenous genes varied considerably in response to both ageing and hydration. Therefore, using the external HMBS was a suitable alternative to evaluate gene expression in these highly contrasting physiological conditions. The reference genes EF1α and GAPDH were the most stable, and could be used for normalisation of qRT-PCR results under specific circumstances.