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 (<i>Allium sativum</i>), 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, <i>eukaryotic translation initiation factor</i> 4<i>α</i> (<i>eIF-4α</i>), <i>actin</i> (<i>ACTIN</i>), <i>tubulin β</i>-7 (<i>TUB7</i>), <i>TAP42-interacting protein of 41 kDa</i> (<i>TIP41</i>), <i>glyceraldehyde-3-phosphate dehydrogenase</i> (<i>GAPDH</i>), <i>SAND family protein</i> (<i>SAND</i>), <i>elongation factor 1 alpha</i> (<i>EF</i>-1<i>α</i>), and <i>protein phosphatase 2A</i> (<i>PP2A</i>) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, <i>ACTIN</i> was the best reference gene for normalizing gene expression. In garlic clove, <i>ACTIN</i> and <i>SAND</i> 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 <i>ACTIN</i> and <i>SAND</i>, 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.