Selection of reference gene in Eucalyptus camaldulensisfor real-time qRT-PCR

Abstract

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), 4C oumarate CoA Ligase (4CL), Cinnamoyl CoA Reductase (CCR )a ndCinnamoyl 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. Materials and methods Samples were collected from three different tissues of E. camaldulensis namely xylem, young leaf and mature leaves and three developmental stages viz one, one and half and three years. The samples were named as X (1-3) for all the xylem samples coming from three developmental stages and Y(1-3) and M(1-3) for young leaves and mature leaves respectively (Figure 1). Total RNA was extracted with RNAqueous Kit (Ambion, Austin TX, USA) following manufactures protocol. cDNA was synthesized from 1 μg of RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) with random hexamer primers. Primers for house keeping genes and lignin biosynthetic genes were designed based on the EST data from Euca