ARS | Publication request: Reference gene selection, primer design and amplification interference of quantitative real-time PCR in tomato and orange subjected to environmental and disease stress

Submitted to: Florida State Horticultural Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 10, 2012
Publication Date: N/A

Technical Abstract: Response of gene expression in the oxylipin pathway to chilling and heating in tomatoes at full ripe stage was investigated. Total RNA was isolated from tomato pericarp tissue, and gene expression of Tomlox A-Tomlox D, HPL and ADH were determined by real-time Polymerase Chain Reaction (PCR) using the SYBR Green PCR kit, and expressed as ''CT in comparison to the reference controls, GAPGH and RPL8. The widely used reference gene GAPGH was more stable than RPL8 in the postharvest stress research. Primer PCR efficiencies of the genes were determined using a 100-fold dilution series of a bulked complementary DNA (cDNA) sample over three dilution points, and the results showed that primers designed using Primer Express 3.0 (Applied Biosystems) had higher efficiency over other primer design programs in the ABI PRISM 7500 Sequence Detection Fast System. For orange research, total DNA was extracted from Huanglongbing infected fruit juice. Citrus cytochrome oxidase (COX) was selected to represent host gene, and 16S rDNA was selected to represent the pathogen bacteria, Candidatus Liberibacter asiaticus (CLas). Amplification of 16S rDNA was inhibited by unknown PCR enzyme inhibitors presented in the DNA extraction. However, the inhibitors were removed by pathing through a silicon spin column. A multiplex qPCR of COX and CLas 16S DNA showed that the amplification of CLas 16S DNA was inhibited by COX in an ABI PRISM 7500 FAST sequence detection system.