Evaluation of reference genes for quantitative RT-PCR in Lolium perenne

Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant development. Partial sequences of nine L. perenne housekeeping genes were obtained by RT-PCR using degenerate primers designed from the corresponding genes in closely related species. Primers for quantitative RT-PCR were designed based on partial sequences. The housekeeping genes were evaluated for their expression stability in different tissues at various stages of development. The analysis found that eEF-1 alpha and eIF-4a were the most stable and beta-TUB was the least stable of the genes tested when all tissues were analyzed together. Analysis by geNorm indicated that the four most stably expressed housekeeping genes (eEF-1 alpha, eIF-4a, 25S rPNA, and GAPDH) should be utilized when normalizing gene expression during plant developmental studies. For root crown tissues at different stages of development, eIF-4a and 25S rRNA were the most stably expressed of the housekeeping genes tested. In leaf tissues, eEF-1 alpha and UBQ5 were the most stably expressed of the housekeeping genes tested. We found that using two housekeeping genes as reference genes is sufficient during RT-PCR gene expression studies when analyzing either root crown or leaf tissues during different stages of development.