Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

Abstract

Real-time RT–PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.

Genome-wide identification and characterization of the aquaporin gene family in Sorghum bicolor (L.)

“…Expression levels of the SbACP2, EIF4A and S/T-PP genes were used as internal controls. These three reference genes are widely used as reference genes in different plant species (Basa et al, 2009;Chandna et al, 2012;Gimeno et al, 2014;Jain et al, 2006;Pinheiro et al, 2011;P.S. Reddy et al unpublished data).…”

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization inIris. lacteavar.chinensisRoots under Cadmium, Lead, and Salt Stress Conditions

“…A number of attempts for reference gene validation have been reported under heavy metal stress in Arabidopsis thaliana [10], soybean [11], cucumber [12], citus [2], and poplar [13] and under salt stress in potato [4], rice [14], tobacco [15], cucumber [16], and Brachypodium distachyon [17]. I. lactea var.…”

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses

“…Short growth cycle plants are limited by low biomass, while plants (especially woody plants) with high biomass and long growth cycles are more able to deal with severe abiotic stress conditions. Only a small number of reference genes have been reported in trees under drought, salt, and heavy metal stress conditions1415161718.…”