Selection of reference gene in Eucalyptus camaldulensis for real-time qRT-PCR

Background Real time quantitative reverse transcription PCR (realtime qRT-PCR) is an established technique for quantification of mRNA and has been extensively used for gene regulation studies in plants. However, there are inherent challenges associated with the technique such as variability of RNA and extraction protocols, different rates of reverse transcription and PCR efficiencies. This demands an accurate method of normalization to obtain reproducible results. Among the various methods, normalization to a reference house keeping gene is the most commonly used method. If inappropriate reference genes are used for normalization, the experimental results can vary significantly leading to false results [1]. Eucalyptus camaldulensis Dehnh. is a widely distributed tree species used for planting in arid and semi-arid areas. The wood is composed of mainly cellulose and lignin and the pathway involved in lignin formation is fairly understood. The lignin biosynthetic genesviz Ferulate 5 Hydroxylase (F5H), 4 Coumarate CoA Ligase (4CL), Cinnamoyl CoA Reductase (CCR) and Cinnamoyl Alcohol Dehydrogenase (CAD) are highly conserved across the tree species and have been utilized as targets for manipulating lignin content. The present study describes validation of a reference gene in various tissue types of eucalyptus and its subsequent use for the expression analysis of lignin biosynthetic genes.