Abstract
BackgroundRT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.ResultsIn this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.ConclusionOur study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.
Highlights
RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies
Several recent studies have scrutinized the stability of commonly known reference genes like 18S ribosomal RNA (18S rRNA), β-actin (ACT), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) used for the quantification of mRNA expression, and have documented that these genes should be used with caution as internal controls, because they showed different behaviors under different experiment conditions [8,9,10,11,12,13]
The specificity of the amplifications was confirmed by the presence of a single band of expected size for each primer pairs in agarose gels following electrophoresis and by the single-peak melting curves of the PCR products
Summary
RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Several recent studies have scrutinized the stability of commonly known reference genes like 18S ribosomal RNA (18S rRNA), β-actin (ACT), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) used for the quantification of mRNA expression, and have documented that these genes should be used with caution as internal controls, because they showed different behaviors under different experiment conditions [8,9,10,11,12,13] The reason for these expressional variabilities may be that the reference genes participate in the basic cell metabolism and take part in other cellular process [14,15]. The selection of the most stable gene or set of genes as internal controls is a critical step to control the variability between samples for quantitative gene expression studies with a sensitive RT-qPCR technique [8]
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