Validating Drought Tolerance Genes

Six candidate genes for drought tolerance were selected to be validated with quantitative real-time PCR (qRT-PCR). Gene-specific (an exon-exon junction) primers were designed by PrimerQuest Tool (Integrated DNA Technologies, IA, United States), and the actin protein gene was used as an internal housekeeping reference for normalization between samples. RNA was extracted with the method described previously, and three biological replications in two separate wells (technical replication) were applied. The cDNA was synthesized by SensiFAS cDNA Synthesis Kit (Meridian Bioscience, United States), and qRT-PCR was performed on ABI 7500 Fast Real Time PCR (Applied Biosystems, CA, United States) using the SensiFAST SYBR Lo-ROX Kit (Meridian Bioscience, United States), following methods described by Wang et al. (2021) (link). The relative fold changes were calculated using the comparative CT method (2^–ΔΔCT). The average value of the two technical replications was considered for each biological replication.

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Nouraei S., Mia M.S., Liu H., Turner N.C, & Yan G. (2022). Transcriptome Analyses of Near Isogenic Lines Reveal Putative Drought Tolerance Controlling Genes in Wheat. Frontiers in Plant Science, 13, 857829.

Publication 2022

PrimerQuest Tool ABI 7500 Fast Real Time PCR SensiFAST SYBR Lo-ROX Kit

Actin Biological Cdna Drought tolerance Exon Gene Meridian Primers Protein gene Real time pcr Replication Synthesis

Corresponding Organization : University of Western Australia

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Variable analysis

independent variables

Six candidate genes for drought tolerance

dependent variables

Relative fold changes in gene expression

control variables

Actin protein gene used as an internal housekeeping reference for normalization between samples

controls

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