Identification and Characterization of reference genes from Sorghum genome for qPCR based expression analysis
Student name: Ms Nikita Gupta
Guide: Dr Sonika Gupta
Year of completion: 2016
Host Organisation: Jawaharlal Nehru University, New Delhi
Supervisor (Host Organisation): Dr Manoj K. Sharma
Abstract: Sorghum bicolor is a C4 grass that belongs to Poaceae “a grass family†and is one of the most economically important cereal crops. It is grown worldwide for food, feed and biomass. In the recent years, Sorghum has emerged as a model system to study, understand and improve C4 crops, and also to gain insights in carbohydrate partitioning. Gene expression studies play an important role in characterising gene function and Quantitative Real-time PCR (qPCR) serves to be one of the robust methods for this purpose. Reference genes are used to normalize the mRNA levels among different samples to rule out the variations caused by technical sample handling. Most often constitutively expressing genes “Housekeeping Genes†are used as for this purpose. It has been found that expression of housekeeping gene may vary among different plant species and therefore identification of species-specific genes whose expression is stable, is critical for qPCR based expression analysis.
Objective of this work is to identify putative reference genes for sorghum based upon the literature survey and evaluate the selected candidates to characterize their expression stability in different plant organs and among various cultivars of sorghum. Based upon the literature survey we have selected five genes (Glyceraldehyde 3-phosphate dehydrogenase, α-actin, eukaryotic initiation factor-5, β-tubulin, Ubiquitin). Gene-specific qPCR primers were synthesized and used to check their expression in seedlings of several sorghum cultivars and various organs like leaf, root and stem of sorghum cultivar M35-1. We have used delta-deltaCt method to compare the relative gene expressions. The selected genes have been ranked according to their expression stability (high to low) in different samples.
Keywords: Q-PCR, Reference Genes, Normalization, Endogenous control
