Quantification of Stress-Induced Mitogen-Activated Protein Kinase Expressional Dynamic Using Reverse Transcription Quantitative Real-Time PCR

Abstract

Although it is generally accepted that signal transduction in plant mitogen-activated protein kinase signaling cascades is regulated via rapid posttranslational modifications, there are also several compelling examples of swift stress induced transcriptional activation of plant MAP kinase genes. A possible function of these fast and transient events is to compensate for protein losses caused by degradation of phosphorylated MAP kinases within stimulated pathways. Nevertheless, there is still need for additional evidence to precisely describe the regulatory role of plant MAP kinase transcriptional dynamics, especially in the context of whole stress stimulated pathways including also other signaling molecules and transcription factors. During the last two decades a reverse transcription quantitative real-time PCR became a golden choice for the accurate and fast quantification of the gene expression and gene expression dynamic. In here, we provide a robust, cost-effective SYBR Green-based RT-qPCR protocol that is suitable for the quantification of stress induced plant MAP kinase transcriptional dynamics in various plant species.

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“…Samples from two plants of the same genotype/treatment combination were pooled and two biological replicates of pooled samples were analyzed per each genotype/treatment combination within the single experiment. RNA extraction, cDNA preparation and qPCR analysis including data evaluation were essentially performed as described before ( Křenek and Smékalová, 2014 ; Křenek et al, 2015 ) and involved two technical replicates in qPCR analysis. The whole RT-qPCR experiment was repeated two times and in total four biological and eight technical replicates were analyzed.…”