Evaluation of Candidate Reference Genes for Gene Expression Normalization in Brassica juncea Using Real Time Quantitative RT-PCR

Ruby Chandna,Naveen C Bisht,Rehna Augustine,Christian Schönbach

doi:10.1371/journal.pone.0036918

Ruby Chandna, Naveen C Bisht + Show 2 more

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https://doi.org/10.1371/journal.pone.0036918

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Journal: PLoS ONE	Publication Date: May 11, 2012
Citations: 195	License type: CC BY 4.0

Affiliation: National Institute of Plant Genome Research

Abstract

The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Highlights

Gene expression analysis is extremely important in many fields of biological research
In the present study, we evaluated the expression stability of 12 candidate reference genes across large number of B. juncea samples in an effort to identify a set of stable reference gene(s) for normalization during gene expression studies
Analysis of expression stability using geNorm and NormFinder revealed that the expression of tonoplastic intrinsic proteins-41 (TIPS-41) and clathrin adaptor complex (CAC) are most stable across variable experimental tissues

Summary

Introduction

Gene expression analysis is extremely important in many fields of biological research. Among the widely used methods to measure the levels of gene expression, real time quantitative reverse transcription PCR (qRT-PCR) represents a suitable technology [1]. Sensitive, and reliable method, qRT-PCR provides a rapid mean towards simultaneous measurement of gene expression across different samples [2]. Since this platform is relatively simple coupled with a high level of sensitivity, qRT-PCR is rapidly being adopted as a standard method for performing in-depth expression analysis of number of target genes. For accurate and reliable analysis of target gene expression, normalization of qRT-PCR data with suitable internal reference gene(s) is required [3]. An ideal reference gene should express at constant level in all tissues and at all developmental stages, regardless of the experimental conditions or treatments [6,7]

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