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-tubul… Show more
“…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.…”
The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy. Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors. In this study, the expression profiles of ten candidate genes, ACT1, ACT2, GAPDH, eIF1, eIF2, α-TUB, β-TUB, TBP, RNA Pol II and RP II, were calculated for a suitable reference gene selection in Paeonia ostii T. Hong et J. X. Zhang leaves under various drought stress conditions. Data were processed by the four regularly used evaluation software. A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one. In addition, the geNorm program provided the optimal choice of two reference gene combination, RNA Pol II and β-TUB, for qRT-PCR normalization in P. ostii subjected to different drought stress levels. Our research provided convenience for gene expression analysis in P. ostii under drought stress and promoted research of effective methods to alleviate P. ostii drought stress in the future.
“…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.…”
The definition of relatively stable expressed internal reference genes is essential in both traditional blotting quantification and as a modern data quantitative strategy. Appropriate internal reference genes can accurately standardize the expression abundance of target genes to avoid serious experimental errors. In this study, the expression profiles of ten candidate genes, ACT1, ACT2, GAPDH, eIF1, eIF2, α-TUB, β-TUB, TBP, RNA Pol II and RP II, were calculated for a suitable reference gene selection in Paeonia ostii T. Hong et J. X. Zhang leaves under various drought stress conditions. Data were processed by the four regularly used evaluation software. A comprehensive analysis revealed that RNA Pol II was the most stable gene and eIF2 was the least stable one. In addition, the geNorm program provided the optimal choice of two reference gene combination, RNA Pol II and β-TUB, for qRT-PCR normalization in P. ostii subjected to different drought stress levels. Our research provided convenience for gene expression analysis in P. ostii under drought stress and promoted research of effective methods to alleviate P. ostii drought stress in the future.
“…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].…”
Selecting an appropriate reference gene is of crucial importance for improving the accuracy of qRT-PCR analyses. In this study, strawberry (Fragaria ananassa) seedlings were subjected to different environmental conditions including heat, cold, drought, salt, white-light, blue-light, and red-light treatments. The expression levels of seven candidate reference genes, including Fa18S, FaGAPDH, FaPIRUV, FaDBP, FaHISTH4, FaACTIN1, and FaACTIN2, in the strawberry leaves were measured by qRT-PCR. Then, four programs (geNorm, NormFinder, BestKeeper, and RefFinder) were employed as tools to evaluate the expression stability of the candidate reference genes. The results showed that the expression stability of the reference genes varied under different conditions. For the cold stress and white-light treatments, FaACTIN2 was evaluated to be the most stable reference gene. FaGAPDH should be used as the reference gene under salt-stress condition and red-light treatment. For the data normalization under drought-stress treatment, FaDBP is the recommended reference gene with the highest expression stability. FaHISTH4 was observed to be the best reference gene for data normalization under heat stress and blue-light treatment. This work provides information on selecting reference genes for accurate gene expression analyses of target genes in strawberry leaves under various abiotic stress and light-quality conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.