Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis

TLDR

Stable reference transcripts are essential for accurate gene expression analyses across development and environmental conditions. The study aims to provide PCR primers and probes for novel reference genes to improve transcript normalization in Arabidopsis. Using Affymetrix ATH1 GeneChip data, the authors identified highly stable genes and designed primers for 22 of them, which were tested on 20 diverse cDNA samples. Hundreds of Arabidopsis genes were identified that are more stable and expressed at lower levels than traditional references, and qRT‑PCR confirmed superior stability for many, including genes encoding protein phosphatase 2A, a coatomer subunit, and a ubiquitin‑conjugating enzyme.

Abstract

Abstract Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.