Evaluation of Candidate Reference Genes for Gene Expression Normalization in <em>Brassica juncea</em> Using Real Time Quantitative RT-PCR

<div><p>The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including <em>ACT2</em>, <em>ELFA</em>, <em>GAPDH</em>, <em>TUA</em>, <em>UBQ9</em> (traditional housekeeping genes), <em>ACP</em>, <em>CAC</em>, <em>SNF</em>, <em>TIPS-41</em>, <em>TMD</em>, <em>TSB</em> and <em>ZNF</em> (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of <em>Brassica juncea</em> has been validated. For the normalization of vegetative stages the <em>ELFA, ACT2</em>, <em>CAC</em> and <em>TIPS-41</em> combination would be appropriate whereas <em>TIPS-41</em> along with <em>CAC</em> would be suitable for normalization of reproductive stages. A combination of <em>GAPDH</em>, <em>TUA, TIPS-41</em> and <em>CAC</em> were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, <em>UBQ9</em> and <em>TIPS-41</em> had the most stable expression. Across five cultivars of <em>B. juncea,</em> the expression of <em>CAC</em> and <em>TIPS-41</em> did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.</p> </div>