Validation of a Rice Specific Gene,Sucrose Phosphate Synthase, Used as the Endogenous Reference Gene for Qualitative and Real-Time Quantitative PCR Detection of Transgenes

2004 ◽  
Vol 52 (11) ◽  
pp. 3372-3377 ◽  
Author(s):  
Jiayu Ding ◽  
Junwei Jia ◽  
Litao Yang ◽  
Haibo Wen ◽  
Chengmei Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reference Gene ◽  
Sucrose Phosphate Synthase ◽  
Pcr Detection ◽  
Specific Gene ◽  
Real Time Quantitative Pcr ◽  
Endogenous Reference Gene ◽  
Endogenous Reference ◽  
Sucrose Phosphate

scholarly journals Validation of a Carnation-Specific Gene, ANS, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR for Carnations

2011 ◽  
Vol 94 (4) ◽  
pp. 1227-1232 ◽  
Author(s):  
Hong Zhu ◽  
Lingxi Jiang ◽  
Shiru Tao ◽  
Heyan Lin ◽  
Jinbin Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Copy Number ◽  
Specific Gene ◽  
Endogenous Reference Gene ◽  
Low Copy Number ◽  
Endogenous Reference ◽  
Pcr Assays

Abstract The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modifed (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplifcation products were obtained with all of them. No amplifcation products were observed with samples from 14 other plant species, which demonstrated that the system was specifc to carnation. The results of Southern blot analysis confrmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR effciency and linearity. Thus, the ANS gene had species specifcity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.

Validation of Artemisia Genus-Specific Gene, GEL4, for Use as an Endogenous Reference Gene in Qualitative PCR Detection of Putative Transgenic Artemisia annua

2013 ◽  
Vol 96 (3) ◽  
pp. 587-592
Author(s):  
Hua Liu ◽  
Lingxi Jiang ◽  
Furong Tan ◽  
Xinghai Zhao ◽  
Heyan Lin ◽  
...  
Keyword(s):  
Reference Gene ◽  
Artemisia Annua ◽  
Intraspecific Variability ◽  
Specific Gene ◽  
Endogenous Reference Gene ◽  
Medical Plant ◽  
Endogenous Reference ◽  
Pcr Method ◽  
Artemisinin Content ◽  
Qualitative Pcr

Abstract Artemisia annua L. is a popular medical plant used against malaria. Transgenic A. annua plants have been developed to increase the artemisinin content using genetic engineering. To develop a reliable PCR method of detecting transgenic A. annua, one Artemisia specific gene, GEL4, was selected and validated as suitable. Qualitative PCR methods were assayed with 16 different A. annua ecotypes and four Artemisia plants, and identical amplified products were obtained. No amplified products were observed when genomic DNA samples were extracted from 15 different plant species. An LOD of 0.05 ng of A. annua DNA was also determined. Furthermore, the results of a Southern blot method showed that the GEL4 gene was in three copies of the six A. annua ecotypes tested. To determine whether there was intraspecific variability in the GEL4 region between different A. annua ecotypes, the GEL4 region corresponding to the 10 A. annua ecotypes was cloned and sequenced. Alignment of the obtained sequences presented a highly conserved amplification product region in 10 A. annua ecotypes. Therefore, it is concluded that the GEL4 gene can be used as an endogenous reference gene of A. annua, and that the qualitative PCR system was reliable for the detection of transgenic A. annua.

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