Abstract
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.
Highlights
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method of quantifying mRNA levels of gene expression [1]
Ten candidates were chosen as reference genes for RT-qPCR analysis
The standard curves indicated that the RT-qPCR amplification efficiency of candidate genes ranged from 92.53% (ARF) to 102.80% (EF1α), and the correlation coefficients varied from 0.991 (COX) to 0.999 (Table 1 and Supplementary Figure S2)
Summary
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method of quantifying mRNA levels of gene expression [1]. Selection of appropriate reference genes is crucial for validating accurate gene expression [2, 3]. Improper reference genes used in data processing may lead to inaccurate and even wrong results [4]. The commonly used reference genes in RT-qPCR analysis are the housekeeping genes because they are usually expressed steadily at mRNA levels in any organs under various conditions [5, 6]. 18S ribosomal RNA (18S rRNA), actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are usually taken as reference genes [5], their mRNAs are not always stable in any cases [7,8,9,10]. Selection and optimization of reference genes are important steps in RT-qPCR experiments
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