Identification of Tps2 Used as an Endogenous Reference Gene in Qualitative and Real-time Quantitative PCR Detection of Flammulina velutipes

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.

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Identification of reference genes for expression analysis by real-time quantitative PCR in drought-stressed soybean

Pesquisa Agropecuária Brasileira ◽

10.1590/s0100-204x2011000100008 ◽

2011 ◽

Vol 46 (1) ◽

pp. 58-65 ◽

Cited By ~ 20

Author(s):

Renata Stolf-Moreira ◽

Eliana Gertrudes de Macedo Lemos ◽

Ricardo Vilela Abdelnoor ◽

Magda Aparecida Beneventi ◽

Amanda Alves Paiva Rolla ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Reference Gene ◽

Expression Analysis ◽

Stable Gene ◽

Expression Stability ◽

Hydroponic System ◽

Mean Values ◽

Real Time Quantitative Pcr ◽

Ct Data

The objective of this work was to validate, by quantitative PCR in real time (RT-qPCR), genes to be used as reference in studies of gene expression in soybean in drought-stressed trials. Four genes commonly used in soybean were evaluated: Gmβ-actin, GmGAPDH, GmLectin and GmRNAr18S. Total RNA was extracted from six samples: three from roots in a hydroponic system with different drought intensities (0, 25, 50, 75 and 100 minutes of water stress), and three from leaves of plants grown in sand with different soil moistures (15, 5 and 2.5% gravimetric humidity). The raw cycle threshold (Ct) data were analyzed, and the efficiency of each primer was calculated for an overall analysis of the Ct range among the different samples. The GeNorm application was used to evaluate the best reference gene, according to its stability. The GmGAPDH was the least stable gene, with the highest mean values of expression stability (M), and the most stable genes, with the lowest M values, were the Gmβ-actin and GmRNAr18S, when both root and leaves samples were tested. These genes can be used in RT-qPCR as reference gene for expression analysis.

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