Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

Abstract

The aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane.

Selection of Stable Reference Genes for Quantitative Real-Time PCR on Paeonia ostii T. Hong et J. X. Zhang Leaves Exposed to Different Drought Stress Conditions

“…Based on the geNorm analysis results, RNA Pol II and β-TUB were stably expressed under different drought stress conditions, which were identified as the most stable reference genes. RNA Pol II and β-TUB were also chosen for internal controls in foxtail millet (Setaria italica L.) [60] and sugarcane (Saccharum officinarum L.) [61] under dehydration stress and drought stress. NormFinder recommended TBP as the most stable reference gene, and RNA Pol II ranked second.…”

Stable Reference Gene Selection for qRT-PCR Normalization in Strawberry (Fragaria × ananassa) Leaves under Different Stress and Light-Quality Conditions

“…However, in another study, the candidate reference gene FaDBP (DNA binding protein) was considered to be the most suitable for normalizing the gene expression data in samples of two strawberry cultivars under drought stress conditions. The candidate reference gene FaHISTH4 (histone H4) showed the highest stability of expression levels under osmotic stress treatments, whereas the traditional reference genes FaGAPDH (glyceraldehyde-3-phosphate dehydrogenase) and Fa18S (18S ribosomal RNA) were ranked as the most unstable genes under all conditions [18]. For strawberry fruit under different light-quality treatment, FaGAPDH offered the most stable expression [10].…”