Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Abstract

Bromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

HMBS is the most suitable reference gene for RT‑qPCR in human HCC tissues and blood samples

“…Also, when performing NormFinder analysis (another tool for calculation of stability), HMBS exhibited the highest stability in tissue samples among the five candidate reference genes (data not shown). In conclusion, HMBS was selected as the most stable reference gene in tissue, serum and serum exosomes based on Bestkeeper, a software that identifies the suitable reference gene ( 39 ). Additionally, NormFinder analysis revealed that HMBS is the most stable reference gene for tissue samples.…”

Selection and validation of suitable reference genes for gene expression studies in Callerya speciosa (Champ. ex Benth.) Schot under different experimental conditions

“…Real-time polymerase chain reaction (RT-qPCR) is easy to perform with high sensitivity and speci city, which provides the accuracy and produces reliable as well as rapid quanti cation results 25,26 , therefore it has emerged as a robust and widely used methodology for gene expression. To date, some plants have been selected for research on RT-qPCR analysis, including Glycyrrhiza 27 , Nitraria tangutorum 20 , Fucus distichus 28 , Bromus sterilis 19 , Allium tuberosum 29 , and Suaeda glauca 30 . In addition, RT-qPCR is widely used in diagnosing diseases, such as preterm birth 31 , diabetic kidney disease 32 , Bronchopulmonary dysplasia 33 .…”

“…For example, in Gossypium hirsutum L. salt stress, UBQ7, GAPDH and EF1A8 were the better reference genes in leaves, while the better reference genes were TUA10, UBQ7, CYP1, GAPDH and EF1A8 in roots 18 . For Bromus sterilis, three developmental stages (2-leaves stage, 3-leaves stage and 4-leaves stage), 18S rRNA and ACCase were the better reference gene 19 . Under ABA treatment, the ideal reference genes of Nitraria tangutorum were EF1-α and His 20 .…”

Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

“…The steps of RT-qPCR are prone to technical noises and variations in the sample preparation. Hence, to nullify these variations, appropriate normalization methods are necessary [ 8 ]. The target gene transcription levels must be normalized with suitable reference gene transcription levels.…”

“…The most commonly used references genes in plant gene expression studies include ubiquitin (UBQ), eukaryotic elongation factor (eEF), α-tubulin (α-TUB), β-tubulin (β-TUB), actin (ACT), ribosomal RNA genes (rRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Acetyl CoA Carboxylase (ACCase) , etc. [ 7 , 8 ]. Although it can be assumed that these genes will have a stable expression in any given condition, numerous studies have provided evidence that their expression levels can vary considerably across plant species, stress conditions, or even developmental stages [ 7 , 11 ].…”