Evaluation of housekeeping gene expression stability in carnation (Dianthus caryophyllus)

Wenjing Yu,Yuan Tao,Liping Luo,Jonathan Hrovat,Ahui Xue,Huolin Luo

doi:10.1080/01140671.2021.1883069

Wenjing Yu, Yuan Tao + Show 4 more

Open Access

https://doi.org/10.1080/01140671.2021.1883069

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Abstract

ABSTRACT The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. Suitable reference gene is an absolute prerequisite for accurate normalisation. In this study, three computational statistical methods before performing RT-qPCR, including geNorm, NormFinder and BestKeeper was used to integrated expression stability evaluations of 12 frequently-used reference genes in Dianthus caryophyllus across different experimental conditions. The results showed that the expression stability of candidate genes varies greatly in different sample pools. The expression of TIP41 (TIP41-like family protein) and UBQ10 (ubiquitin10) was relatively stable under different experimental conditions, while CYP (cytochrome P450) and TUA (a tubulin) could act as reliable internal controls in different tissues. The reliable reference genes under treatment of hormone, heavy metal, salt, heat, cold, flooding and drought were EF1α (elongation factor 1 alpha)/TIF5A (translation initiation factor 5A), UBQ10/18S (18S Ribosome RNA), UBQ10/CYP, TUB (β-tubulin gene)/TIP41 (TIP41-like family protein), TIF5A/EF1α, TUB/TIP41 and TIP41/PP2A (protein phosphatase 2A). Some common housekeeping genes, such as ACT (actin gene) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), exhibited unstable expression patterns. This was the first systematic study on the stability of internal reference genes of selection of reference genes in Carnation.

Highlights

The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability
EF1α, TIP41 and UBQ10 were considered the most appropriate reference genes when all samples were put together, while UBQ10 was most stable in exogenous hormone treatments
TUB and UBQ10 can be used as reliable internal control genes under stress, while CYP and TUA can act as reliable internal controls in different tissues

Summary

Introduction

The real-time quantitative reverse transcription PCR (RT-qPCR) is widely used for gene expression analysis, owing to its advantages of high specificity, sensitivity and repeatability. Real-time quantitative reverse transcription PCR (RT-qPCR) is a method developed from traditional PCR technology that has strong specificity, sensitivity and high throughput for the quantification of nucleic acids [2, 8]. It has been widely used in molecular biology, medicine and other basic research fields, especially in gene function studies, strain identification, and the inspection and quarantine of animal- and plant-derived pests and diseases[21, 25, 27]. ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (EF1α), and ubiquitin-conjugating enzyme (UBC) are commonly used internal reference genes in plant gene expression analysis experiments [17]

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