Identification and Validation of Different Housekeeping Genes for Real Time Rt-Pcr in Plants

Investigations of gene expression patterns of a gene or set of different genes in eukaryotic cells at particular stage or conditions require appropriate set of internal controls for accurate quantitative data analysis. These internal controls or reference genes are essential for ensuring the cell viability. Housekeeping gene/s (HKG) has been commonly used as reference genes for the normalization of gene expression data due to their presumed stable and constitutive expression in living organisms. The selection of appropriate internal control gene is a critical step for the gene expression studies by qRT-PCR due to variability in the stable expression of candidate HKGs in different organisms, different organs/tissues of same organism or most imporatantly due to different experimental conditions or environmental conditions for the same sample. In this study, we challenged the consensual thinking that all HKGs are reliable controls for expression studies through detailed investigation of set of potential reference genes suitable for gene expression analysis of Ficus carica (dicot), Chenopodium album (dicot) and Mentha spicata (dicot) after treating with different experimental conditions of abiotic stresses. We identified and sequenced three isoforms of actin (actin, β-actin and actin α), two isoforms of tubulin gene (β-tubulin and β-tubulin-1), GAPDH, EFα1, ubiquitin and 18S rRNA genes from these three plants and checked their validity as good internal control genes. Gene specific primers of above mentioned genes were designed from the conserved regions of similar genes from other plants to amplify genomic DNA followed by sequencing. After confirmation of right products from each plant using several bioinformatics tools, all of these sequences were submitted to genebank. Initially the similarities of these newly isolated genes with other known homologues or orthologues were determined. Alignments and phylogenetic trees have shown high levels of conservation in the genes among diverse set of selected monocots and dicots, as expected. In order to validate the utility of these newly isolated genes as internal controls, we treated the above mentioned plants with various abiotic stresses including heat, metal, cold, drought, salt and growth hormones. The respective transcripts were amplified and sequenced for further confirmations. We could hardly observed significant difference in the expression of these genes by semi- quantitative RT-PCR as expected. For quantitative validations, we used all of the above candidate internal controls to study the differences in their expression by relative quantitative real-time to validate the best gene or set of genes for the normalization of target genes in these plants. Different reference genes of C. album, F. carica and M. spicata showed somewhat variable expression under one or the other type of abiotic stress treatment suggesting; 1) Different experimental conditions can variably affect the stability of these internal control genes. 2) β-tub, EFα1, Ubq and Act-α were identified as the most stable genes under cold, drought, heat, metal and salt stress in C. album. In F. carica, 18S rRNA, β-tub and Act were three stable genes under cold, drought, salt and growth hormone stress. Similarly, GAPDH, β-tub, β-tub-1 and EFα1 were the stable internal control genes under drought, cold, heat and salt stress in M. spicata respectively. Furthermore, the gene stability index was generated for these selected HKGs which facilitated the selection of more than one internal control gene for further verifications of gene expression studies of these or any other related plant species.