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Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

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Published on: October 27, 2017

Selection and validation of reference genes in alfalfa based on transcriptome sequence data.

Wenna Fan¹, Yaqi Shi², Pengfei Shi²

¹Animal Science and Technology College, Henan University of Science and Technology, Luoyang, 471003, Henan, China. chou0516@163.com.

Scientific Reports

|February 21, 2025

Summary

This summary is machine-generated.

This study identifies optimal internal reference genes for RT-qPCR in alfalfa under various abiotic stresses. Traditional genes like GAPDH and Actin are not ideal; specific genes such as UBL-2a and Ms.33,066 are recommended for accurate gene expression analysis.

Keywords:

Alfalfa Reference gene Selection Transcriptome sequence

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Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

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Area of Science:

Plant molecular biology
Genomics and transcriptomics

Background:

Quantitative gene expression analysis using RT-qPCR requires reliable internal reference genes for accurate normalization.
Traditional reference genes (GAPDH, Actin) may not be stable across diverse experimental conditions, particularly abiotic stresses in plants like alfalfa (Medicago sativa L.).

Purpose of the Study:

To identify and validate the most stable internal reference genes for RT-qPCR in alfalfa under five different abiotic stress conditions (drought, alkali, high temperature, low temperature, and acid).
To evaluate the suitability of commonly used reference genes (GAPDH, Actin) and novel candidate genes for accurate gene expression normalization in alfalfa.

Main Methods:

Candidate reference genes were selected from alfalfa transcriptome data (162 RNA-seq datasets).
Expression stability of 10 candidate genes was assessed using RT-qPCR under drought, alkali, high temperature, and low-temperature stresses.
Gene stability was evaluated using multiple algorithms and software, including GeNorm, Normfinder, Bestkeeper, ΔCt method, and RefFinder.

Main Results:

GAPDH and Actin were found to be unsuitable as reference genes under various abiotic stresses in alfalfa.
Optimal single reference genes identified: UBL-2a (alkali), Ms.33,066 (drought), and Actin (high/low temperature).
Optimal reference gene combinations were determined for alkali (MS.65,463 and UBL-2a) and drought (MS.65,463 and UBL-2a) stresses, with further combinations suggested for high and low temperatures.

Conclusions:

The study provides crucial data for selecting appropriate reference genes in alfalfa gene expression studies under abiotic stress.
Novel reference genes and combinations are recommended, offering improved accuracy over traditional genes for RT-qPCR analysis in alfalfa.
Further validation of optimal reference genes under acid stress is warranted.