Genome-Wide Constitutively Expressed Gene Analysis and New Reference Gene Selection Based on Transcriptome Data: A
Jiaping Zhao1, Fan Yang1,2, Jinxia Feng1
1State Key Laboratory of Tree Genetics and Breeding, Institute of New Forestry Technology, Chinese Academy of Forestry, Beijing, China.
Frontiers in Plant Science
|November 23, 2017
Summary
This study introduces a new method to find stable reference genes from transcriptome data, improving gene expression analysis accuracy. The findings show commonly used house-keeping genes are often unreliable, recommending new candidates for better data normalization.
Area of Science:
- Plant Molecular Biology
- Bioinformatics
- Gene Expression Analysis
Background:
- Transcriptome datasets are valuable for understanding organismal biology but contain untapped potential for analytical tool development.
- Accurate normalization of gene expression data is crucial for reliable transcriptome analysis across diverse conditions.
- Commonly used house-keeping genes often lack the required stability for accurate gene expression normalization.
Purpose of the Study:
- To develop and describe a methodology for identifying candidate reference genes from high-throughput sequencing data.
- To improve the accuracy of quantitative real-time polymerase chain reaction (RT-qPCR) by establishing better standards for data normalization.
- To evaluate the suitability of newly identified reference genes compared to commonly used house-keeping genes in *Populus bejingensis*.
Main Methods:
- Analysis of transcriptome data from *Populus bejingensis* inoculated with *Botryosphaeria dothidea*.
- Expression stability analysis using coefficient of variance (CV) and maximum fold change (MFC) thresholds.
- RT-qPCR validation of candidate reference genes and comparison with commonly used house-keeping genes.
Main Results:
- Identified 729 stably expressed poplar transcripts (1.11%) with CV < 20% and MFC < 2.0.
- Demonstrated that 70 out of 72 commonly used house-keeping genes were not stably expressed, with issues like multiple isoforms and non-specific amplicons.
- Newly identified genes (e.g., SSU_S8e, LSU_L5e, 20S_PSU) exhibited higher expression stability than most traditional house-keeping genes.
- Normalization using four new reference genes yielded results comparable to Cufflinks output for differential expression analysis.
Conclusions:
- Proposed a pipeline for deriving robust reference genes from transcriptome data, emphasizing unique transcripts, constitutive expression, and specific CV/MFC/expression level criteria.
- The developed methodology provides a reliable alternative for normalizing transcriptome data, essential for cross-platform and cross-species integration.
- This approach enhances the precision of gene expression studies and establishes better standards for data analysis in plant biology.
Keywords:
Botryosphaeria dothideadifferential expressionexpression stabilityhigh-throughput sequencinghouse-keeping geneintegrate analysisinternal controlpoplar
