Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation

Methylation of cytosine plays an important role in epigenetic gene regulation in vertebrates and higher plants [1]. In contrast to animals, where methylated cytosine residues are primarily observed within the symmetrical CpG dinucleotide, plants display cytosine methylation in any DNA context, including symmetric CG and CHG (where H = A, T or C) and asymmetric CHH [2]. Over the past few decades, four major approaches have been used for distinguishing the epigenetic mark 5-methylcytosine (5mC) from unmethylated cytosine. These include methods based on isochizimer restriction endonucleases, bisulphite conversion of DNA, immunoprecipitation and mass spectrometry [3,4]. Bisulphite conversion of DNA, originally developed by Frommer et al. [5], involves treatment of DNA with sodium bisulphite, where under optimized conditions unmethylated cytosine is converted to uracil, whilst methylated cytosine (both 5mC and 5-hydroxymethylcytosine) remains unchanged. DNA sequence changes resulting from bisulphite conversion can then be detected by a variety of methods, including PCR amplification, followed by DNA sequencing where in the original uracil residues are reported as thymine. The primary advantage of this technique is that it provides base-pair resolution of methylation patterns, which is particularly useful in plants for distinguishing between the different cytosine sequence contexts [6]. Following a number of substantial improvements based on the original protocol, bisulphite sequencing is now accepted as the gold standard for detecting changes in DNA methylation [3]. The combination of bisulphite conversion and next-generation high-throughput sequencing has recently provided powerful tools for revealing DNA methylation patterns on a genome-wide scale [7-10].Although bisulphite-based methods are reasonably accurate and reproducible in comparison with other methods, successful detection is dependent on the complete bisulphite conversion of all unmethylated cytosine into ura