Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

Abstract

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L

“…GAPDH has been reported as the most stable reference gene under PEG and cold stress in I. lactea var. chinensis 37 , but it showed unstable expression under various environmental conditions in garlic plants 46 and under PEG and NaCl treatments in Glehnia littoralis 47 . In our study, IgGAPDH was the most stable reference gene across all flower developmental stages in ‘00246’.…”

Validation of Reference Genes for Quantitative PCR in Johnsongrass (Sorghum halepense L.) under Glyphosate Stress

“…However, there is no universal gene that satisfies the requirements, and so the reference gene of choice becomes dependent on factors such as the sample and tissue type, the experimental conditions, and the integrity of the sample [ 25 ]. Several groups have searched for suitable reference genes for expression analysis under different conditions in plants, including weeds [ 26 , 27 , 28 , 29 ]. A number of recent studies have focused on the stress under herbicide treatment for weeds [ 21 , 30 , 31 , 32 , 33 ].…”

Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis of Flowering Stages and Different Genotypes of Iris Germanica L.

“…chinensis 34 and at different flower developmental stages in tree peony 16 , but it showed unstable expression under various environmental conditions in garlic plants 42 . In our study, IgGAPDH was the most stable reference gene across all flower developmental stages in '00246'.…”