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

被引:40
作者
Martin, Ruth C. [1 ]
Hollenbeck, Vicky G. [1 ]
Dombrowski, James E. [1 ]
机构
[1] ARS, USDA, Natl Forage Seed Prod Res Ctr, Corvallis, OR 97331 USA
关键词
D O I
10.2135/cropsci2007.10.0597
中图分类号
S3 [农学(农艺学)];
学科分类号
0901 ;
摘要
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.
引用
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页码:1881 / 1887
页数:7
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