Selecting Reference Gene From Public Microarray Data And Validating By QRT-PCR In Zea Mays

Maize is one of the most important grain crops in the world and the largest food crop in the China. Microarray, a powerful tool for researches of gene function, gene identification, disease treatment, gene expression in development stage, which can detect the gene expression with high-throughput, has been widely applied in the research of gene expression in maize. qRT-PCR is widely used for its high sensibility, high specificity, and good reproducibility. And gene expression can be quantified rapidly and efficiently by qRT-PCR. Reference gene is a class of gene which expressed relatively stable in organizations and cells. By using reference genes in qRT-PCR, effects of differences among samples in RNA quality, reverse transcription efficiency and other man-made factors on qRT-PCR results can be eliminated to correct the expression of target genes. So far until now, there is no optimal reference gene which can stay the stable expression regardless of the changing experimental conditions, In previous researches of gene expression in maize, the used reference genes were conventional reference genes; however, the high varitable expression in different cells, organizations and experimental conditions,one of the defects of the conventional reference genes, had been discovered in the researches in the recent years. In this study, novel reference genes were mined for the microarray database to study the maize gene expression in different conditions, and this is critical to the identification of genes which can improve maize grain field, quality, stability and resistance.In this study, novel maize reference genes were screened out by collecting microarray data from the published database. By means of qRT-PCR detection and reference gene stability estimation using software geNorm and NormFiner, the most stably expressed reference genes and their suitable numbers were screened out. The results are as follows.1.In this study,143candidate maize reference genes were screened out via analysis of microarray data of different physical processes of maize, this provides tremendous available candidate genes for maize researches in the level of mRNA. These candidate reference genes need to be verified in experiments to further validate their stability so as to provide references for future research in maize mRNA fields.2.The qRT-PCR primer design was optimized. Primers designed by NCIB Primer-Blast and Primer3Plus in this procedure would aligned via NCBI Blast firstly, the being aligned through BLAST in Maize GDB database to acquire the specific electronical product, and using PrimerRankTool to evaluate the primer. This procedure with high standardization and strong specificity greatly improved the successful rate of primer design, accelerated the experimental process, and saved the cost.3.Novel reference genes of maize were screened by microarray firstly in this study.7of the14candidate reference genes were annotated while7were unknown. These reference genes were validated to stably expressed under stress such as low temperature and drought; and the four genes NM001112080, NM001112147, NM001137886and NM001156648, were identified as the most stable genes in this study via the analysis of gene stability estimation software geNorm and NormFinder.