High-Throughput Plant Gene Expression Analysis by 384-Format Reverse Transcription-Quantitative PCR for Investigating Plant Iron Homeostasis

Abstract

Analysis of plant gene expression is important in determining iron (Fe) homeostasis gene functions during plant development or in response to biotic and abiotic factors. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has many advantages. It is fast, inexpensive, accurate, and reproducible in any lab. Furthermore, RT-qPCR can be scaled up to study several genes of interest in many biological samples from any organism. We hereby provide a straightforward protocol on RT-qPCR analysis using a 384-well format for large-scale gene expression studies on Fe-regulated responses. The protocol highlights in detail, the steps ranging from choice and design of qPCR analysis, collection of plant material and RNA preparation, cDNA synthesis, set up of qPCR and run, thorough analysis of qPCR run data, and display of multiple gene expression data for convenient interpretation.