Abstract
Real-time quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for gene expression studies. However, choosing the appropriate reference gene is essential to obtain reliable results for RT-qPCR assays. In the present work, the expression of eight candidate reference genes, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), UBC (ubiquitin-conjugating enzyme), UBQ (polyubiquitin), ACT (actin), β-TUB (β-tubulin), APT1 (adenine phosphoribosyltransferase 1), and 18S rRNA (18S ribosomal RNA), was evaluated in Achyranthes bidentata samples using two algorithms, geNorm and NormFinder. The samples were classified into groups according to developmental stages, various tissues, stresses (cold, heat, drought, NaCl), and hormone treatments (MeJA, IBA, SA). Suitable combination of reference genes for RT-qPCR normalization should be applied according to different experimental conditions. In this study, EF1-α, UBC, and ACT genes were verified as the suitable reference genes across all tested samples. To validate the suitability of the reference genes, we evaluated the relative expression of CAS, which is a gene that may be involved in phytosterol synthesis. Our results provide the foundation for gene expression analysis in A. bidentata and other species of Amaranthaceae.
Highlights
Real-time quantitative polymerase chain reaction (RT-qPCR) has become one of the most efficient and powerful techniques to study molecular biology analysis of gene expression and is widely used because of its reproducibility, accuracy, quantity, and sensitivity in gene expression analysis
We evaluated the stability of eight candidate reference genes (GAPDH, 18S rRNA, UBQ, EF1α, UBC, β-TUB, APT1, ACT) for normalization across set of biological samples representing A. bidentata under different experiments
In order to verify the specificity of the all primer pairs for these candidate reference genes and one target genes, agarose gel electrophoresis (3%) and melting curve analysis were carried out using RT-qPCR
Summary
Real-time quantitative polymerase chain reaction (RT-qPCR) has become one of the most efficient and powerful techniques to study molecular biology analysis of gene expression and is widely used because of its reproducibility, accuracy, quantity, and sensitivity in gene expression analysis. RT-qPCR is a powerful technique, the results of RT-qPCR data analysis are often affected by different variables, such as RNA purity, RNA quantity, DNA contamination, PCR amplification efficiency, and reverse transcription efficiency (Zhu et al, 2013) To control these variables, it is essential to select one or more suitable reference genes as the commonly applied method for normalizing RT-qPCR data (Guénin et al, 2009; Chen et al, 2011; Kundu et al, 2013). Traditional housekeeping genes that are universally expressed in all cells are often used as reference genes (Xu et al, 2011), such as GAPDH, 18S rRNA, ACT, UBQ, EF1-α, β-TUB, and UBC (Wang et al, 2014; Xiao et al, 2014).
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