Internal Reference Gene Selection under Different Hormone Stresses in Multipurpose Timber Yielding Tree Neolamarckia cadamba

Deng Zhang,Kunxi Ouyang,Jingjian Li,Chunmei Li,Buye Li,Xiaoyang Chen

doi:10.3390/f11091014

Abstract

Neolamarckia cadamba, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL) including 43 genes. The results showed that FPS1, RPL, and FBK were the most stable reference genes across all of the tested samples. In addition, the expression of NcEXPA8, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in N. cadamba.

Highlights

Real-time quantitative PCR (RT-qPCR) is the preferred method for the validation of high-throughput or microarray results and for determining gene expression levels due to its good reproducibility, high sensitivity, accurate quantitation, and fast response [1,2,3]
We report the validation of housekeeping genes to identify the most suitable internal reference genes for normalization of qPCR data obtained among different tissues of N. cadamba under different hormone stresses
A total of 415 N. cadamba UniGenes with high sequence identity (E-value ≤ 1 × 10−10 ) corresponding to 23 A. thaliana housekeeping gene families downloaded from the TAIR10 database were found in the stem database (Supplementary S1)

Summary

Introduction

Real-time quantitative PCR (RT-qPCR) is the preferred method for the validation of high-throughput or microarray results and for determining gene expression levels due to its good reproducibility, high sensitivity, accurate quantitation, and fast response [1,2,3]. In order to acquire reliable and repeatable results, the selection and systematic validation of suitable reference genes as internal controls is an essential prerequisite in RT-qPCR normalization for every specific experimental condition [5]. Several algorithms, such as geNorm [12], NormFinder [13], BestKeeper [14], and ∆Ct [15], have been well developed to validate the most stable reference gene(s) from a series of candidate genes under a given set of experimental conditions

Methods

Results

Discussion

Conclusion