Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze).

Xinyuan Hao,Yajun Yang,Xinchao Wang,David Horvath,Wun Chao,Bin Xiao

doi:10.3390/ijms151222155

Abstract

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions.

Highlights

Gene expression analysis is an important approach, providing insight into the genetic and developmental mechanisms in biological research
To identify suitable reference genes for normalizing Quantitative real-time polymerase chain reaction (qRT-PCR) tea plant gene expression analysis suitable for the expression analysis in several different experimental conditions, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of traditional Arabidopsis reference gene family members encoding glyceraldehyde 3-phosphate dehydrogenases (GAPDHs), elongation factors (EFs), ubiquitins (UBQs) and ubiquitin carrier proteins (UBCs), stably expressed genes identified from whole-genome GeneChip studies that encode clathrins, MONENSIN SENSITIVITY1 (MON1 or SAND), tonoplast intrinsic proteins (TIPs) and polypyrimidine tract-binding proteins (PTBs) [27], and three housekeeping gene currently used in tea plant research that encode 18S rRNAs, actins and tubulins
To identify stable reference genes for gene expression studies in tea plant, 11 candidate reference genes and two target genes were investigated by qRT-PCR using six series of samples

Summary

Introduction

Gene expression analysis is an important approach, providing insight into the genetic and developmental mechanisms in biological research. Quantitative real-time polymerase chain reaction (qRT-PCR) has become a critical tool for rapid and reliable quantification of gene transcript levels due to its simplicity, specificity, and sensitivity [1,2,3]. A successful qRT-PCR assay relies on many factors, including the integrity of purified RNA, reverse transcription efficiency, primer design, detection chemistry selection, and data analysis [4,5]. Normalizing results with a stably-expressed internal reference gene is a simple and popular method for controlling error in qRT-PCR, and is generally preferred over other normalization strategies based on sample size, total RNA quantification, and spiked control molecules [7]. The selection and validation of endogenous reference genes for qRT-PCR in each species is necessary

Results

Discussion

Conclusion