Abstract
BackgroundThe selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.ResultsThe expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.ConclusionsWe recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.
Highlights
The selection of stable and suitable reference genes for real-time quantitative Polymerase chain reaction (PCR) (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions
We evaluated 16 reference genes to establish their suitability as control genes for normalization and identified a set of genes that are suitable for quantitative gene expression analysis by Reverse transcription (RT)-qPCR in C. glabrata following fluconazole treatment
The three ribosomal RNA subunits RDN5.8, 18S ribosomal RNA (RDN18), and 25S ribosomal RNA (RDN25) were the most stable reference genes, with cycle threshold (CT) Change values less than 0.5, while UBC13, Phosphoglycerate kinase (PGK1), and UBC7 were relatively stable with CT Change values of only around 1.5
Summary
The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The most commonly used reference genes, including β-actin, cyclophilin, GAPDH, tubulin, and 18S and 28S ribosomal RNAs, have shown variable expression levels in different cells and tissues under different conditions, and they are unsuitable for normalization purposes owing to large measurement error [6,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33]. It is no longer acceptable to arbitrarily select any reference gene for normalization; it must be demonstrated that the reference gene of choice is suitable for the experiment in question
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