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

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

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: GmB-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 GmB-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.

Saved in:
Bibliographic Details
Main Authors: STOLF-MOREIRA, R., LEMOS, E. G. de M., ABDELNOOR, R. V., BENEVENTI, M. A., ROLLA, A. A. P., PEREIRA, S. dos S., OLIVEIRA, M. C. N. de, NEPOMUCENO, A. L., MARCELINO-GUIMARÃES, F. C.
Other Authors: RENATA STOLF-MOREIRA, UNESP Jaboticabal; ELIANA GERTRUDES DE MACEDO LEMOS, UNESP Jaboticabal; RICARDO VILELA ABDELNOOR, CNPSO; MAGDA APARECIDA BENEVENTI, UFRGS; AMANDA ALVES PAIVA ROLLA, UEL; SELMA DOS SANTOS PEREIRA, UEL; MARIA CRISTINA NEVES DE OLIVEIRA, CNPSO; ALEXANDRE LIMA NEPOMUCENO, CNPSO; FRANCISMAR CORREA MARCELINO, CNPSO.
Format: Artigo de periódico biblioteca
Language:English
eng
Published: 2011-03-24
Subjects:Estabilidade de expressão, Expressão gênica, RT-qPCR, Soja, Biotecnologia, Deficiência hídrica, Soybeans, Gene expression, Plant-water relations, Agricultural biotechnology,
Online Access:http://www.alice.cnptia.embrapa.br/alice/handle/doc/882641
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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: GmB-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 GmB-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.

Similar Items