ARS | Publication request: Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 9, 2008
Publication Date: March 1, 2009
Citation: Dombrowski, J.E., Martin, R.C. 2009. Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress. Plant Science. 176:390-396.

Interpretive Summary: Quantitative real-time (RT)-PCR provides an important tool for analyzing gene expression if proper internal standards are used. Currently there are limited molecular resources publicly available for forage and turf grass analysis. In order to develop molecular tools for gene expression analysis in grasses, we identified evaluated nine reference genes for use in real-time quantitative RT-PCR in the model grass species Lolium temulentum (Darnel grass) under 7 different forms of abiotic stress. We identified specific housekeeping genes to be used for normalization of data during expression analysis for grass undergoing stress and that utilization of the two most stable two housekeeping genes as reference genes is sufficient for proper RT-PCR gene expression analyses. The information presented in this manuscript provides important and valuable information to researchers studying gene in expression in forage and turf grasses.

Technical Abstract: Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR for use in abiotic stress studies in L. temulentum. Partial sequences of nine L. temulentum 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 these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to different forms of abiotic stress. The analysis found that eEF-1a and UBQ5 were the most stable and ACT11 was the least stable of the genes tested. Analysis by geNorm indicated that the two most stably expressed housekeeping genes (eEF-1a and UBQ5) should be utilized and are sufficient for normalization of gene expression during stress related studies in L. temulentum.