Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

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Date: Aug. 31, 2012
From: PLoS ONE(Vol. 7, Issue 8)
Publisher: Public Library of Science
Document Type: Report
Length: 9,725 words
Lexile Measure: 1610L

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Author(s): Xiaoyang Zhu, Xueping Li * , Weixin Chen, Jianye Chen, Wangjin Lu, Lei Chen, Danwen Fu

Introduction

Gene expression analysis is an important step to understand the roles of genes in developmental and cellular processes, such as the signaling and metabolic pathways [1]. Real-time reverse transcription PCR (RT-qPCR) has emerged as the most widely used method to quantify changes in gene expression profiles in response to different environmental conditions. It has shown important attributes such as accuracy, precision and relative ease of use due to its speed, sensitivity and specificity [2], [3]. Nevertheless, to accurately quantify gene expression, several experimental variations, such as quality and amount of starting material, presence of inhibitors in different sample materials, primer design, and RNA extraction and retro-transcription efficiencies, should be taken into account [4]. Therefore, selection of an appropriate normalization strategy is of crucial importance for the acquisition of biologically meaningful data. Among several methods proposed so far [4], [5], the use of one or more reference genes is currently the preferred method of normalization [6]. An ideal reference gene should be expressed at a constant level across various conditions and its expression is assumed to be unaffected by experimental parameters [7], [8]. Moreover, the reference gene and the target gene should have similar ranges of expression in the samples to be analyzed [9]. Genes involved in basic metabolism and maintenance of the cell, e.g. [beta]-actin, glyceraldehyde-3-phosphatede hydrogenase (GAPDH ), ribosomal subunits and ubiquitin are commonly used as reference genes [10], [11], [12]. However, several reports have demonstrated that there are no universally applicable reference genes with an invariant expression, and that the using of unstable reference gene will lead to inappropriate biological data interpretation [8], [13], [14]. Thus, there is an urgent need to systematically evaluate the stability of potential reference genes for every particular experimental condition prior to their use in RT-qPCR normalization. Meanwhile, several algorithms, such as geNorm [15], NormFinder [16], BestKeeper [17], qBasePlus [18], and RefFinder [19] have been well developed to validate the most stable reference gene(s) from a panel of potential genes or candidate genes under a given set of experimental conditions [20], [21].

Recently, a growing number of reference gene validation attempts have been reported for plants, such as Brachypodium distachyon [22], potato [23], sugarcane [24], rice [25], [26], [27], Petunia hybrid [28], soybean [29], tomato [30], [31], wheat [32], barley [33], grape [34], poplar [35], coffee [36], Arabidopsis thaliana [13], cucumber [37], chicory [38], pisumsativum [39], Swingle Citrumelo [40], Populus [41], Peanut [42], lichi [43], tobacco [44], banana [20] and citrus [45]. However, there have been no reports on the suitability of reference genes for RT-qPCR studies of differential expression of genes in papaya.

Papaya (Carica papaya L. ) is the only species within the genus Carica and the most commercially important species within the family Caricaceae [46] and it has been widely cultivated in tropical and subtropical lowland regions for its nutritional benefits and medicinal applications. Papaya is also the first perennial transgenic fruit variety for commercial application of the...

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Gale Document Number: GALE|A498243615