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

Renata Stolf-Moreira,Francismar Corrêa Marcelino-Guimarães,Amanda Alves Paiva Rolla,Magda Aparecida Beneventi,Alexandre Lima Nepomuceno,Maria Cristina Neves De Oliveira,Selma Dos Santos Pereira,Eliana Gertrudes De Macedo Lemos,Ricardo Vilela Abdelnoor

doi:10.1590/s0100-204x2011000100008

Abstract

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.

Highlights

Soybean has been the subject of many studies carried out to understand and quantify the processes that interfere with crop production in challenging environments (Popp et al, 2003)
Organ, the Gmβ‐actin and GmRNAr18S genes were considered the best references for this trial, because the RQ values of the target gene normalized with these two reference genes remained constant (Table 2)
Primer amplification efficiency for each reaction with the reference genes was determined from the slope of the exponential phase of amplification by RT‐qPCR, within an experiment designed to compare organ‐specific differences in gene expression (Table 1)

Summary

Introduction

Soybean has been the subject of many studies carried out to understand and quantify the processes that interfere with crop production in challenging environments (Popp et al, 2003). The quantification of mRNAs is usually achieved by northern blotting or by ribonuclease protection assay (RPA) (Suzuki et al, 2000). These methods are less precise than RT‐qPCR (Bustin, 2000), which has emerged as an important technique to compare the expression profiles of target genes in several species, tissues and treatments, and to validate high‐throughput gene‐expression profiles (Crismani et al, 2006). A reference gene should have stable expression in different organs, developmental stages, and environments. According to previous works on the best reference genes for transcription normalization in plants, the most reliable are those constitutively expressed and involved in basic cellular processes, such as protein and sugar metabolism and maintenance of cell structure (Cruz et al, 2009)

Objectives

Methods

Results