Traditional and novel references towards systematic normalization of qRT-PCR data in plants

The selection of appropriate controls is the key requirement for target gene normalization through quantitative real-time PCR. The consistent control is selected via a gene validation approach that includes several classic and novel housekeeping references. Our aim in this paper is to present major achievements, largely concerned with finding an invariable internal control in different plant species under a range of experimental conditions. The conflicts in gene normalization using classical references and their replacement with novel references are discussed as leading subject. This review also accentuates different methods used for the amplification of reference genes with special importance on ortholog/paralog approaches, and multi-transcript targeting. Some other issues are also discussed such as the use of quantitative real-time PCR in different areas of plant science for target gene normalization, and some areas where it might be a useful technique in unraveling other issues.