Selection of appropriate reference genes for quantitative real-time reverse transcription PCR in Betula platyphylla under salt and osmotic stress conditions.

Ziyi Li,Chao Wang,Huijun Lu,Zihang He,Xiaoyu Ji,Yucheng Wang,Mayank Gururani

doi:10.1371/journal.pone.0225926

Ziyi Li, Chao Wang + Show 5 more

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https://doi.org/10.1371/journal.pone.0225926

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Abstract

Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.

Highlights

Quantitative real-time reverse transcription polymerase chain reaction has become a common technique to detect gene expression levels in the field of molecular biology because of its speed, high efficiency, labor-saving, and sensitivity
Agarose gel electrophoresis showed that single PCR products could be amplified from the 11 candidate reference genes (S1 Fig), indicating that the primers designed by Primer 5.0 software had strong specificity and fulfilled the requirements of Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) primers
The mean cycle threshold (Ct) value can illustrate the abundance of the transcripts from the 11 candidate reference genes (Table 1), which can be used to analyze the stability of reference genes in different experimental samples

Summary

Methods

In this experiment, open pollinated northeast white birch (B. platyphylla) seeds from the Northeast Forestry University were planted in plastic pots with mixture of turf peat and sand (v/v 3:1), in a growth house where the conditions kept at 70–75% relative humidity, 400 μmol m-2s-1 light intensity, a 16 h light/8 h dark photocycle at 25±2 ̊C. The 8-week-old birch plants grown in soil were watered with a solution of 2 L 200 mM NaCl or 2 L 300 mM mannitol (distilled water served as the control) for 3, 6, 12, 24, and 48 h, respectively. The root, stem, and leaf tissues from 6 birch seedlings were collected after each treatment. All the samples were placed at −80 ̊C after freezing in liquid nitrogen for further study.

Results

Discussion

Conclusion